S1

1

2

Supporting Information for 3

�4

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Daniel Girma Mulat1, Alastair James Ward

1, Anders Peter S. Adamsen

1, Niels Vinther Voigt

2, Jeppe 7

Lund Nielsen3, Anders Feilberg

1* 8

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13

*Corresponding author: 14

Anders Feilberg; phone: +45 30896099; e0mail: af@eng.au.dk�15

16

17

18

19

20

21

22

23

24

25

26

27

19 pages, 6 figures, 2 tables, 8 sections were included in the supporting materials. 28

S2

������������������������� �����������������������������������29

Acetoclastic methanogenesis (AM): 30

*CH3COO

0 + H2O →

*CH4 + HCO3

0 ∆G°´= 031.0 kJ mol

01 (S1) 31

Syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis (SAO0HM): 32

*CH3COO

0 + 4H2O → H

*CO3

0 + HCO3

0 + 4H2 + H

+ ∆G°´= +104.1 kJ mol

01 (S2) 33

H*CO3

0 (or HCO3

0) + 4H2 + H

+ → *CH4 (or CH4) + 3H2O ∆G°´= 0135.6 kJ mol

01 34

(S3) 35

(asterisks represent the carbon of the methyl group in acetate) (1, 2) 36

37

�������������38

���������������39

A schematic picture of the experimental set0up for the MIMS measurement of anaerobic digestion and 40

standard dissolved gas is presented in Figure S1. The quadrupole mass spectrometer (Balzers QMG 41

420) was equipped with a membrane pump for rough pumping and a turbo pump for generating high 42

vacuum pumping down to ~1006

mbar. The analytes were ionized by electron ionization (70 eV) and 43

the ions were separated according to their mass to charge ratio (�01) by a quadrupole mass filter and 44

finally detected with a secondary electron multiplier. QuadstarTM version 6.02 software was used for 45

acquisition of data. Mass spectra were acquired at 5 s amu−1

. Characteristic ions of each compound 46

were monitored in multiple ion detection (MID) mode. 47

N21

2

3

Exhaust gas

reservior

MFC

QMS

Cold trap

MFC

MIMS

probe

Magnetic

stirrer

Anaerobic reactor

in a water bath

CO2/CH4

�48

S3

�������� MIMS measurement set0up for calibration of standard dissolved gases and for measurement 49

of dissolved gases in anaerobic reactor. During calibration of dissolved gases in water, the three gas 50

lines (�, � and �) were connected. During the MIMS measurement of anaerobic digestion products, the 51

two gas lines (� and �) were disconnected. MFC is mass flow controller; QMS is quadrupole mass 52

spectrometer. 53

54

The anaerobic reactor system was constructed by connecting three gas lines to a gas0tight serum bottle 55

(see Figure S1). Gas line � was made by connecting stainless steel and capillary tubing as follows. One 56

end of the 0.32 cm o.d. stainless steel tubing was connected to the high vacuum section of the mass 57

spectrometer through a bellows0sealed valve (Swagelok, Denmark) for safety purpose and the other end 58

was connected to 0.32 cm o.d. capillary tubing. The stainless tubing was bent to a U0shape and kept in 59

a 1 L wide0mouth Dewar flask (Sigma Aldrich, Denmark) filled with dry ice (~ 080°C) to make a cold 60

trap system. The other end of the 0.32 cm o.d. capillary tubing was connected to a membrane probe. 61

The membrane probe was made from 127 Lm thick silicon membrane (SIL0TEC Sheeting, Technical 62

Products Inc., USA) inlet and supported by a thin perforated stainless steel plate. Before connecting the 63

capillary tubing to the membrane probe, a hole was drilled in a butyl rubber stopper (30 mm o.d., 64

Apodan Nordic, Denmark) and the capillary tubing was inserted directly in this. A serum bottle 65

containing fermentation broth was sealed with the butyl rubber stopper and secured with an aluminum 66

crimp. The stated MIMS setup was used for measurement of anaerobic digestion process. 67

68

A small modification was made to the MIMS setup during preparation of standard gas solution. 69

Additional two gas lines (� and �) were made (see Figure S1). Two holes were drilled in the butyl 70

rubber stopper and two lines of PTFE tubing (0.32 cm o.d.) were inserted directly in these. Gas line � 71

was constructed by connecting one of the PTFE tubing from the serum bottle through a three0way 72

connector to two mass flow controllers (EL0FLOW®

Select, Bronkhorst) for adding a specified flow of 73

CO2/CH4 and N2 gases2 One mass flow controller (MFC) was connected to a gas mixture cylinder (60 74

vol% CH4 and 40 vol% CO2) and the other MFC was connected to a gas cylinder of N2 (purity > 99.99 75

vol%) with PTFE tubing (0.64 cm o.d.). Gas line � was based on submerging the other end of the PTFE 76

tubing from the serum bottle into a beaker filled with water for removing exhaust gas. 77

78

S4

Two types of MIMS measurements were employed: 79

(i) MIMS calibration:� Aqueous standard solutions of carbon dioxide and methane at different 80

concentrations were prepared in deionized water from the standard gas mixture CH4 (60 vol%) and CO2 81

(40 vol%). A N2 gas (purity > 99.99 vol%) was used for dilution. The dilution was carried out by 82

adjusting the volumetric flow of the component gases in the mixture by using a MFC2 A gas mixture 83

(CO2 and CH4) and N2 gas with defined concentration (vol%) was bubbled into a serum bottle (500 84

mL) filled with 200 mL of deionized water. The gas was continuously bubbled into water until the 85

concentration of dissolved gas was stable. The stabilization took 40045 minutes and it was monitored 86

with MIMS by recording the characteristics ions of methane at �01 15 and carbon dioxide at �01 44287

(ii) MIMS measurement of anaerobic digestion: A serum bottle (5000mL) containing a fermentation 88

solution was quickly transferred from the incubation chamber to a water bath (52°C) for MIMS 89

measurement. The serum bottle was uncapped and the MIMS probe was immediately submerged into 90

the fermentation broth and the bottle was capped again. During capping and uncapping the bottle, N2 91

gas was used for flushing across the mouth of the bottle. The effectiveness of N2 flushing for avoiding 92

the diffusion of air into a reactor was tested by incubating two parallel sets of unlabeled acetate 93

reactors. One set of reactors was capped all the time until the end of the experiment. The other set was 94

used for MIMS measurement where the bottles were capped and uncapped during the measurement as 95

explained earlier. The acetate degradation rate and methane production profile of both sets of control 96

reactors were similar (data not shown). N2 flushing across the mouth of the bottles is an effective 97

technique in order to avoid the diffusion of air into the fermentation bottle. Sufficient mixing during 98

MIMS measurement was provided with a magnetic stirrer. After every MIMS measurement, the 99

membrane probe was first rinsed with acidified water (0.1 M H2SO4) and then deionized water in order 100

to avoid contamination of the inlet system and to provide a constant background signal. After the 101

MIMS measurement, the serum bottle was kept in the incubation chamber until the next measurement 102

day. 103

104

������������������105

The volume of a produced gas was measured using an acidified water displacement method at room 106

temperature and atmospheric pressure. Samples of headspace gas were taken using a gas tight syringe 107

with a needle through a septum and transferred into a 20 mL headspace vial. The compositions of CH4 108

S5

and CO2 in the headspace samples were analyzed using Agilent technologies 7890A gas 109

chromatograph equipped with a thermal conductivity detector and an Agilent technology GC sampler 110

80. Methane and carbon dioxide were isolated using Alltech’s CTR I stainless steel column. It is 111

essentially a column within a column which was packed with different materials: outer column (1.8 m 112

x 0.64 cm) was packed with an activated molecular sieve and inner column (1.8 m x 0.32 cm) was 113

packed with a porous polymer mixture. The carrier gas was helium at 30 mL/min. The temperatures of 114

injection port, column oven and detector were set at 110, 40 and 150 °C, respectively. 115

116

Liquid samples for VFA analysis were withdrawn with a syringe equipped with a needle through a 117

septum and its pH was measured immediately. The liquid samples (1.000 g) were first acidified with 4 118

mL of 0.3 M oxalic acid containing the internal standard dimethylpropanoic acid, then centrifuged at 119

4,500 rpm for 12 min and filtered through a 0.45m GHP membrane. Finally an aliquot of the 120

supernatant solution was transferred into a vial. The concentrations of volatile fatty acids were 121

determined by Agilent Technologies 7890A gas chromatograph equipped with flame ionization 122

detector (FID). A polar phase capillary column, HP0INNOWax (30m x 0.25 mm x 0.25 Lm), was used 123

for separation. Helium was used as a carrier gas at 1.8 ml/min flow rate. The analyses were performed 124

using a temperature programme: 5 min at 100°C, a linear gradient from 1000120°C at the rate of 10 125

°C/min, 5 min at 120°C, a linear gradient from 120°C to the final temperature of 220°C at the rate of 126

30°C/min and final hold at

220°C for 3 min. The temperatures of injection port and detector were set at 127

285°C and 300°C, respectively. 128

129

�����������������130

Liquid samples for proteome analysis were collected from all reactors at the middle of the incubation 131

experiment and stored in a freezer (~ 020 °C) until the analysis. Proteins were extracted followed by 132

tryptic digestion as described elsewhere (3) and mass spectrometry analysis by an automated LC0ESI0133

MS/MS with an UltiMate 3000 RSLCnano system on0line coupled to a Q Exactive mass spectrometer 134

via a Nanospray Flex ion source (Thermo Fisher Scientific). The resulting mass spectra were used to 135

search for protein homologs using an in0house Mascot database search engine and the NCBI and the 136

UniProt database as described in details elsewhere (4). 137

138

S6

����������������������139

The characteristic ion signal intensities were used for calculating the isotopic composition of methane 140

and carbon dioxide. Peaks at �01 45 and 44 were regarded as signals from 13

CO2 and 12

CO2, 141

respectively. A peak at �01 17 was regarded as a molecular ion for 13

CH4. A peak at �01 15 includes the 142

signals from 12

CH3+ and

13CH2

+. According to the fragment ions of

13CH4 mass spectrum, the signal 143

from 13

CH2+ represented 22.8% of the peak intensity of �01 17 (data not shown). The signal intensity of 144

12CH3

+ at �01 15 was corrected by subtracting the calculated signal intensity for

13CH2

+ from the total 145

signal intensity at �01 15. The corrected signal intensity at �01 15 represents 12

CH3+ which is a 146

fragment ion of 12

CH4. An example of MIMS data correction and presentation is given in Table S1. The 147

data was taken from MIMS measurement of [2013

C] acetate and [U013

C] acetate reactors on the first day 148

of the incubation time. 149

150

The inoculum used to incubate [2013

C] acetate substrate was not free from inorganic carbon species 151

(CO2 (dissolved), HCO30 and CO3

20). The sum of these inorganic carbon species in a solution of the 152

inoculum were represented as background total inorganic carbon (TIC). The background TIC of the 153

inoculum was calculated according to equation S4 using the CO2(dissolved) measured with MIMS, 154

carbonates (HCO30 and CO3

20) equilibrium constants and pH of the inoculum (5). 155

CO������� CO���� ����� ���������������������

����� � (S4) 156

157

The MIMS results of the [2013

C] reactor are presented after correcting the contribution of the 158

background TIC to �01 44 (12

CO2) and �01 15 (12

CH4) signals of the [2013

C] sodium acetate reactor. The 159

MIMS measurement data for [U013

C] acetate reactor was used to make the TIC correction. The 160

unlabeled products (12

CO2 and 12

CH4) during the degradation of [U013

C] acetate were considered as the 161

background TIC that comes from the inoculum and they were subtracted from the MIMS values of 162

12CO2 and

12CH4 measured during the degradation of [20

13C] acetate. An example of TIC correction is 163

given in Table S1. 164

165

S7

166

�� ���� Isotopic distribution of methane and carbon dioxide from 13

C labeled acetate 167

168

Ion current

Substrate �01 15 (12

CH4) in nA

�01 17 (13

CH4)

in nA 13

CH4 (atom%)

Actual

13CH2

+

subtracteda

Background

TIC0correctedb Actual

Background

TIC0correctedc

13CH3COO

0 4.0 2.4 1.4 7.0 83.7

13CH3

13COO

0 3.3 1.0 9.9

Ion current

Substrate �01 44 (12

CO2) in nA

�01 45 (13

CO2)

in nA 13

CO2 (atom%) 13

CO2 (atom%)/13

CH4(atom%)

Actual

Background

TIC0correctedd Actual

Background

TIC0corrected

Background

TIC0corrected 13

CH3COO0 49 45.1 0.7 1.6 0.02

13CH3

13COO

0 3.9

169

a.� The signal from 13

CH2+ represented 22.8% of the peak intensity of �01 17. 170

b.� The ion current at �01 15 from [U013

C] acetate was regarded as arising from the background 171

production of unlabeled CH4. 172

c.� The percentage of 13

CH4 to total methane calculated from background TIC0corrected 12

CH4 and 173

actual 13

CH4 values. 174

d.� The ion current at �01 44 from [U013

C] acetate was regarded as arising from the background 175

production of unlabeled CO2. 176

177

����������������������������������178

The production of CH4 and /or CO2 from acetate depends on the metabolic pathways. During 179

acetoclastic methanogenesis, methane is formed from the methyl group carbon atom in acetate and 180

carbon dioxide is formed from the carboxyl group carbon atom (6). In synthrophic acetate oxidation 181

pathway, the carbons of both the groups in acetate are converted to carbon dioxide (7, 8). If 182

acetoclastic methanogenesis is taking place, 1 mole of 13

CH4 and 1 mole of 12

CO2 will be produced 183

from 1 mole of 13

CH3COO0. When SAO is taking place, 1 mole of

13CO2, 1 mole of

12CO2 and 4 moles 184

of H2 will be produced from 1 mole of 13

CH3COO0. Since there is no extra source of H2 available to the 185

system, only 1 mole of carbon dioxide is reduced by 4 moles of H2 via HM and the other 1 mol of 186

carbon dioxide is remained unconsumed. Both 13

CO2 and 12

CO2 can be reduced to 13

CH4 and 13

CH4, 187

respectively, with different proportion according to their rate constants. As 12

CO2 reacts slightly faster 188

S8

than 13

CO2 during the reduction of carbon dioxide (i.e., 12!/

13! = 1.065 on average at thermophilic 189

condition) (9, 10), the production of 12

CH4 is estimated to be slightly higher than 13

CH4 at the given 190

time point. A schematic representation of calculated mass balance of 13

CH4, 12

CH4, 13

CO2 and 12

CO2 by 191

assuming 50% methane production from acetoclastic methanogenesis and the remaining 50% from 192

SAO0HM is shown in Figure S2. In this mass balance calculation, the kinetic isotope effect (12!/

13! = 193

1.065) was included. The background TIC was not included in the mass balance calculation since TIC0194

corrected MIMS data is used to quantify the contribution of SAO to methane production. Similar mass 195

balance calculation was reported by Hori �� �$2(11) but we expanded it by including the kinetic isotope 196

effect. 197

S9

�� !"#$%&

'()��%

HMHM

50% AM 50% SAO

13CH3COOH

=50 mM

13CH3COOH=100 mM

13CH3COOH

=50 mM

12CO2(AM)

=50 mM12CO2(SAO)

=50 mM

H2

=4x50=200 mM

13CO2(SAO)

=50 mM

SAOAM

198

199

200

S10

������ ���A schematic representations to determine the mass balance of 12

CH4, 13

CH4, 12

CO2 and 201 13

CO2. Herein an example of a mass balance is provided when 100mM 13

CH3COOH was degraded by 202

50% acetoclastic methanogenesis and 50% synthrophic acetate coupled to hydrogenotrophic 203

methanogenesis. HM is hydrogenotrophic methanogenesis; AM is acetoclastic methanogenesis; SAO is 204

synthrophic acetate oxidation; atom% of 13

C labeled substrate is {13

C substrate/(13

C substrate + 12

C 205

substrate)} x 100, for e.g. 13

CO2 (atom%) ={13

CO2/(13

CO2+12

CO2)} x 100; 12

CO2 (H2eq) and 13

CO2 206

(H2eq) are 1 mol 12

CO2 and 13

CO2 equivalent that can be reduced by the available 4 mol H2 via HM , 207

respectively, i.e. only 50 mM of 12

CO2 and 13

CO2 can be reduced by the available 400 mM of H2; KIE 208

is kinetic isotope effect; 12!/

13!=1.065 is the ratio of the rate constants of

12CO20derived methane to 209

13CO20derived methane; HM,KIE is methane0derived via hydrogenotrophic methanogenesis including 210

kinetic isotope effect. 211

�� ���������212

For measurement of 13

C labeled methane (13

CH4) at �01 17, the main interference comes from the 213

fragment ion of water (OH+). The silicone membrane used in our experiment is permeable to low0214

molecular mass volatiles, water and gases. A cold trap was used to condense the water vapor before it 215

reaches the ionization source of the mass spectrometer. A Dewar flask containing dry ice (~ 080°C) was 216

placed between the membrane probe and mass spectrometer in order to trap water vapor. The cold trap 217

was very effective in condensing water that otherwise interferes with 13

CH4 measurement. The signal 218

intensity of water measured at �01 18 was five0folds reduced when the cold trap was used (Figure S3). 219

The reduction of the amount of water vapor in the mass spectrometer reduced the pressure inside the 220

mass spectrometer (data not shown). The provision of high vacuum inside the mass spectromer enables 221

accurate propagation of ion trajectories which in turn improves the detection limit and the reliability of 222

measurement (12). 223

224

�225

0

5

10

15

20

25

30

0 50 100 150 200

*���������#�$&

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S11

��������� Implementation of the cold trap (operated at ~80°C) caused a significant reduction of the 226

water vapor content (as monitored by �01 18), which otherwise permeate through the silicone 227

membrane. The lines represent the first 95 scans in the absence of cold trap and the last 90 scans in the 228

presence of cold trap. When cold trap was used, the data points until the attainment of stable MIMS 229

reading was excluded. That is why a sharp drop between the top left line and the bottom right line was 230

observed. 231

232

"������������������������233

The aim of the sample temperature test was to investigate the effect of membrane temperature on the 234

operating pressure of the MIMS system and performance of the silicone membrane. The temperature of 235

deionized water as a sample was increased step by step from room temperature to 52.5°C. The choice 236

of this temperature range was based on the fact that most of biogas production from organic waste is 237

conducted under either mesophilic (~ 35°C) or thermophilic (~ 52°C) conditions. In our study, 238

anaerobic digestion was conducted in the incubation chamber held at 52°C. While the MIMS 239

measurement was conducted, the temperature of the reactor was controlled by a water bath held at 240

52°C. Constant temperature was maintained during MIMS measurements to avoid process instability 241

due to temperature fluctuation and to stabilize the MIMS measurements, as temperature affects the 242

MIMS signals (see also below). 243

244

Figure S4 shows changes of pressure inside the mass spectrometer with an increase in temperatures of 245

water sample from room temperature up to 52.5°C. The pressure hardly changed when temperature 246

increased from room temperature up to 29°C. However, it showed a linear three0fold increase as the 247

temperature increased from 29052.5°C. Moreover, the background intensity of both CO2 and CH4 248

signals increased by three0fold (data not shown). Higher sample temperature causes the expansion of 249

membrane micropores, which leads to increase in diffusivity of the analyte through the membrane. As a 250

consequence of higher analyte flow into the vacuum section of the mass spectrometer, the partial 251

pressure inside the mass spectrometer increases (13). In our temperature study, the total pressure in the 252

ionization chamber of the mass spectromer is lower than the recommended safety limit (~ 3.0 x 1005

253

mbar). Therefore, it can be concluded that safe operation of a MIMS system is achievable for a typical 254

sample temperature range (30052°C). 255

S12

256

������ �"� The pressure of ion source (mbar) of the mass spectrometry as a function of silicone 257

membrane temperature (°C). 258

259

(�*��������������������260

When calculating response times for polymer membranes, usually 90% or 50% response times are 261

taken. In our study, we defined the response time as the time required to observe a 90% increase of 262

signal intensity of the ion. Response time vary with length (L) of the probe tube and inversely with the 263

radius (r) of the probe (cm). The 90% response time, �90% is given by (14): 264

�90% = kL2r01

(S5) 265

where k is a constant. The value of k is in the order of 1x1005

and varies with temperature and 266

properties of membrane (15).�267

268

Figure S5 shows the MIMS response time for the measurement of dissolved carbon dioxide and 269

methane in a fermentation reactor containing substrate0free inoculum and pure water. At time zero the 270

sample was changed from pure water to substrate0free inoculum and the MIMS was scanned in MID 271

mode for 4.5 minutes. It can be seen from Figure S5 that the signals of both CO2 and CH4 were 272

increased to 90% maximum in just 1 min and level off for 4.5 min. Then the membrane probe was 273

immediately rinsed with deionized water and dipped inside a reactor containing deionized water. The 274

arrow indicates the change from substrate0free inoculum back to pure water. The signal intensities of 275

both analytes decreased to 10% in just 1 min and then levels off. Our result was in accordance with 276

literature (16) where the rise time (10090% of maximum signal response) is equal to or slightly shorter 277

0

1

2

3

4

5

6

7

8

25 29 42 46 50 52.5

+������#�),-� ��&

���������#. &

S13

than fall time (decrease in signal response from 90010%). The fast rise and fall time obtained in our 278

study shows the capability of MIMS technique for online and onsite monitoring of dissolved carbon 279

dioxide and methane in real time. 280

281

282

283

�������(�MIMS measurement of (a) dissolved methane (�01 15) and (b) carbon dioxide (�01 44) 284

directly inside reactors containing substrate0free inoculum and deionized water. The arrow indicates the 285

change from substrate0free inoculum back to pure water. 286

287

-�%�������������������/����%*%��288

The determination of the concentration of hydrogen at �01 2 in our reactor was obscured by the high 289

concentration of dissolved methane (about 1.4 mM) and very low concentration of dissolved hydrogen 290

ranged from 0.5 to 3 XM in the digester (17). Due to high energetic electron (70 eV) used for the 291

ionization of the analytes, methane (CH4) molecules fragment to H2+ ion and detected at �01 2. The 292

contribution of methane to the signal at �01 2 was determined by purging methane at different 293

concentration into deionized water. As shown in Figure 6S, a linear relationship was observed (r2 = 294

0.995) when �01 2 signal was plotted versus �01 15 signal (methane). Due to the high background 295

signal from methane at �01 2 and the expected extremely small concentration of hydrogen at steady 296

anaerobic condition, the ion abundance of hydrogen at �01 2 could not be determined accurately in our 297

experiment. However, the hydrogen concentration of anaerobic digester both at steady state organic 298

loading rate (OLR) and increased OLR was reported by some authors (18020). In these published 299

0

500

1000

1500

2000

2500

0 4.5 9

*�������������(�#��)��$

)

��������#���&

(a)

0

50

100

150

200

0 4.5 9

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(b)

S14

works, the calibration curve for quantifying hydrogen and the interference of methane at �01 2 were not 300

reported. The reported hydrogen data might be overestimated due to the interference from fragment ion 301

of methane. It seems that the concentration of hydrogen in an anaerobic digester can only be 302

determined accurately by MIMS in a few condition, for instance, at high OLR that leads to high 303

concentration of hydrogen and with appropriate correction for the interference caused by the fragment 304

ion of methane. 305

��306

307

�������- The fragment ion of methane at �01 2 divided by �01 18 (water) and its linear dependence 308

with the ion intensity of �01 15 (methane) divided by �01 18 (water). 309

0�%������������������310

The use of ion0current ratio between 13

C and 12

C isotopes of CO2 and CH4 in our study significantly 311

reduces errors associated with the drift in mass spectrometer and membrane inlet system as well as 312

slight fluctuations in sample temperature and permeability of water vapor. We have considered two 313

major sources of uncertainty, which arises from isotope ratio measurement with MIMS and the 314

calculation of quantifying methanogenesis pathways as follows. 315

(i) Uncertainty of MIMS measurement of isotopic distribution of CO2 and CH4: 316

The MIMS measurement was conducted for 4 minutes in each anaerobic reactor. The maximum 317

response signal was attained in one minute and an average of data points within the last three minutes 318

of MIMS measurement was used to assign the signal intensity of the individual measured ion. The 319

uncertainty associated with taking the average signal intensity was 2.5%, 1.3%, 1.3% and 1.3% for �01 320

15 (12

CH4), �01 17(13

CH4), �01 44(12

CO2) and �01 45(13

CO2), respectively. 321

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0 2 4 6 8

*�������������,��,����1������

*�������������(,��,����1��������

S15

(ii) Uncertainty of quantifying SAO pathways as a function of MIMS data of 13

CO2 (atom%)/13

CH4 322

(atom%) using equation 1: 323

Equation 1 was derived considering the kinetics isotope effect during the reduction of carbon dioxide to 324

methane. The average value of 12!/

13! = 1.065 was used to account the kinetic effect (9, 10). Previous 325

literatures showed that the 12!/

13! for thermophilic hydrogenotrophic methanogens was in the range of 326

1.04 to 1.09 (9, 10). The uncertainty associated with the use of 12!/

13! = 1.065 on average for deriving 327

equation 1 was 4.9%. 328

329

The propagated uncertainty associated with quantification of SAO (%) using equation 1 as a function 330

of MIMS data of 13

CO2(atom%)/13

CH4(atom%) was calculated from both uncertainty components 331

listed above and it was estimated to be 5.9%. 332

333

1�%��� ���������������������334

The resulting mass spectra were used to identify and taxonomically attribute individual peptides to 335

,�#����� and ��#����. At least two peptides with similar identities were required for proper 336

identification. The proteome analysis showed that a total of 527 proteins were identified with a wide 337

range of functional categories and representing 18 phyla, 30 classes and 107 genera. The bacterial 338

proteome constituted 74.5% of the proteins with a relative high diversity whereas the majority of the 339

remaining proteins affiliated to ��#���� (Table S2). We found only a few fungal and plant proteins in 340

the sample. 341

342

343

344

345

346

347

348

349

350

351

S16

�� �����Distribution of protein sequences analyzed in this study 352

�353

+����2 ����23���2� +������

�#���+�#����� 2.3

�#���+�#����� 2.1

�#���+�#����-�� 0.2

�3����#�� 1.1

,�#�����-����0(�$���+�group 5.7

,�#�����-���� 4.4

(�$���+� 1.1

%����+�#������ 0.2

(�$���-���04����#���#��+��group 0.4

(�$����$�5� 0.9

(�$-�$���� 0.2

(�$����$�5� 0.4

"�������#��+�� 0.4

(���+�#����� 0.2

�������+�#����� 0.2

6�+��+�#�����0�#�-�+�#�����group 0.6

6����#���� 30.9

,�#�$$� 8

($�����-�� 19.7

������$����#��� 0.4

�������#���� 2.8

6���+�#����� 0.9

7$�#����#���� 1.1

7�����+�#����� 16.5

�$���������+�#����� 5.3

,���������+�#����� 2.8

8����������+�#����� 5.7

��$��������+�#����� 2.5

���$�������+�#����� 0.2

.����#������ 3.4

.����������� 7

.��������� 7

"���������� 0.8

Uncultured bacteria 2.5

S17

�����#������ 23.3

*�����+�#����� 0.4

*�������#��+�� 23

*�����#�$$� 0.2

*�����#�����#�$�� 0.8

*�����#�$$��� 8.7

*�������$$�� 1.3

*�����$��� 0.6

*������$��� 0.6

*��������#�� 10.8

�����#������ 0.4

(�-�-����Korarchaeum 0.4

"������#������ 0.2

Uncultured Archaea 1.5

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

S18

References 378

379

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S19

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432