Supplemental Information: Table S1. Overview of Reactions

Supplemental Information:

Table S1. Overview of reactions examined in this study. ΔG values were obtained from Thauer et al., 1977.

Reaction Equation ∆G°' (kJ/reaction)*

Acetogenic reactions

– – + 1 Butyrate + 2 H2O → 2 Acetate + H + 2 H2 +48.3 Sulfate-reducing reactions

– 2– – – + 2 Butyrate + 0.5 SO4 → 2 Acetate + 0.5 HS + 0.5 H –27.8

2– + – 3 4 H2 + SO4 + H → HS + 4 H2O –151.9

– 2– – – 4 Acetate + SO4 → 2 HCO3 + HS –47.6 Methanogenic reactions

- + 5 4 H2 + HCO3 + H → CH4 + 3 H2O –135.6

– – 6 Acetate + H2O → CH4 + HCO3 –31.0

Syntrophic butyrate conversion

7 Butyrate– + 0.5 HCO – + 0.5 H O → 2 Acetate– + 0.5 CH + 0.5 H+ –19.5 3 2 4 Table S2. Overview of all the enrichment slurries fed with butyrate and the total amounts of the reactants consumed and products formed during the enrichment period. The enrichment slurries consisted of sediment either from sulfate zone (SZ), sulfate-methane transition zone

(SMTZ) or methane zone (MZ) and were incubated at 25°C or 10°C, with 3 mM, 20 mM or without (-) sulfate amendments along the study. Slurries with * were presented in the butyrate conversion graphs and used for molecular analysis.

Sediment Incubation Reactants (µmol/slurry) Products (µmol/slurry) Slurry Code Treatment zone temperature ( C) Butyrate Sulfate Acetate Sulfide Methane *B1 - 25 35085 313 37497 717 63347 B2 - 25 38415 312 29386 508 64830 2- B3 20 mM SO4 25 36896 37342 109911 39836 1030 2- *B4 20 mM SO4 25 36816 35382 74468 33996 3252 SZ *B5 - 10 22092 351 24613 460 19853 B6 - 10 24609 319 31487 364 12978 2- *B7 20 mM SO4 10 19587 15551 33563 17064 93 2- B8 20 mM SO4 10 22414 15282 35718 18012 0 2- *B1 3 mM SO4 25 33451 5500 10151 7479 73569 2- B2 3 mM SO4 25 33748 8020 30128 9107 69486 2- *B3 20 mM SO4 25 40754 45716 45255 38991 4183 2- B4 20 mM SO4 25 41405 47734 46877 42512 3727 SMTZ 2- B5 3 mM SO4 10 26272 6463 35148 7321 32045 2- *B6 3 mM SO4 10 28158 6531 37574 7268 32483 2- *B7 20 mM SO4 10 29226 35141 31129 36984 35 2- B8 20 mM SO4 10 33078 31697 41505 32413 1015 2- B1 20 mM SO4 10 26059 18632 35892 14784 2956 2- *B2 20 mM SO4 10 29488 34963 33448 38500 0 2- *B3 20 mM SO4 25 54945 41587 91742 30618 5792 2- B4 20 mM SO4 25 56743 54643 84195 39675 587 MZ *B5 - 25 45588 358 43022 261 105926 B6 - 25 45829 782 29932 487 97903 B7 - 10 18145 610 12727 380 27090 *B8 - 10 16225 601 29455 424 8417 Table S3. The number of reads per sample generated by Pyrosequencing for Bacteria and HiSeq Illumina sequencing for Archaea. ENV: Original Sediment Sample

Origin Slurry Bacterial reads Archaeal reads

ENV 8787 9120 B4 12393 88555 Sulfate zone B1 18687 121246 B7 12007 61722 B5 12194 89283 ENV 3202 18696 B1 3758 75708 Sulfate-methane transition zone B3 5167 47520 B6 5443 42599 B7 7198 58464 ENV 10903 30848 B3 15094 39087 Methane zone B5 11718 136909 B2 11686 52551 B8 10891 94124

Table S4. Relative abundance (HPLC/MS mass chromatogram area, summed by IPL head group per gram sediment dry weight) of all IPLs identified in Aarhus Bay sediment and the enrichment slurries inoculated with this sediment. SZ: Sulfate zone; SMTZ: Sulfate Methane Transition Zone; MZ: Methane zone; Sediment depth: 120 cm = sulfate zone, 135-165 cm = sulfate-methane transition zone, 180-225 cm = methane zone.

Origin Sample Mhex PA DH Betaine DMPE PE PG OL GlcA PC TMO PI LPC B1 0 0 0 0 0 1946176 0 0 0 0 0 0 2954044 B4 0 0 7594944 0 0 1983971 0 0 0 2832247 0 0 849552 SZ B5 0 0 11499437 0 0 4455602 0 0 0 0 0 0 1204249 B7 0 0 0 0 0 10303739 17291589 0 0 1499265 0 0 0 B1 39551686 0 12878869 0 0 12799850 1060523 0 35097613 8812082 0 0 590925 B3 0 0 5197585 0 0 5120743 11400411 1174897 0 0 0 0 0 SMTZ B6 0 0 12235720 0 0 10403673 47667216 0 0 31353022 0 0 711956 B7 0 0 0 0 0 1494608 24104131 0 0 0 0 0 0 B1 0 0 485170 0 0 17021650 18855340 0 0 757859168 15574565 0 16962049 B2 0 68529 0 0 0 1153218 8106805 0 0 0 0 467142 0 B3 0 0 1648930 0 0 2230226 8895759 0 0 0 0 0 1088251 B4 0 0 0 0 0 0 3354888 0 0 110589668 17064261 0 23535905 MZ B5 737203 0 21381687 11195317 2801118 0 0 0 0 12704704 1724058 0 2048403 B6 0 613445 16440021 0 0 4403271 0 0 0 0 0 0 0 B7 0 0 0 0 0 0 0 0 0 0 0 0 0 B8 0 365393 6954652 0 0 1906084 0 0 0 0 0 139512 401081 120 cm 0 531101 0 0 0 1207772 0 0 0 0 0 1131887 0 135 cm 0 443604 0 0 0 0 0 0 0 0 0 0 0 150 cm 0 552721 0 0 0 808333 0 0 0 0 0 0 0 Sediment 165 cm 0 738732 0 0 0 0 0 0 0 0 0 0 0 depth 180 cm 0 1255773 0 0 0 0 0 0 0 0 0 0 0 195 cm 0 373889 0 0 0 0 0 0 0 0 0 0 0 210 cm 0 473870 0 0 0 2551160 0 0 0 0 0 0 0 225 cm 0 247824 0 0 0 0 0 0 0 0 0 0 0 Sulfate & Sulfide (mM) 0 5 10 15 20 0

50 Sulfate zone

100

Sulfate-methane 150

Depth(cm) transition zone

200 Methane zone

250 0.0 0.5 1.0 1.5 2.0 2.5 Methane (mM)

sulfate (mM) sulfide (mM) CH4 (mM)-GC2 CH4 (mM)-GC1

Figure S1. Depth profiles of sediment pore water sulfate, sulfide and methane for Station M1, in Aarhus Bay, Denmark. Methane-GC1 and Methane-GC2 stands for methane concentrations retrieved from two different gravity corers, gravity corer 1 and 2, respectively. 100% Tenericutes - Acholeplasmatales TM6 Spirochaetes - Spirochaeta Spirochaetes - PBS-18 Spirochaetes - MSBL2 Spirochaetes - LK-44f 90% Gammaproteobacteria - Thiotrichaceae Gammaproteobacteria - Piscirickettsiaceae Gammaproteobacteria - Pseudomonadaceae Gammaproteobacteria - Moraxellaceae Gammaproteobacteria - Oceanospirillaceae Gammaproteobacteria - Ectothiorhodospiraceae 80% Gammaproteobacteria - Shewanellaceae Gammaproteobacteria - Pseudoalteromonadaceae Gammaproteobacteria - Colwelliaceae Gammaproteobacteria - Alteromonadaceae Gammaproteobacteria - Alteromonadales Gammaproteobacteria 70% Epsilonproteobacteria - Helicobacteraceae Epsilonproteobacteria - Campylobacteraceae Deltaproteobacteria - Sva0485 Deltaproteobacteria - Desulfuromonadaceae Deltaproteobacteria - Desulfuromonadales Deltaproteobacteria - Desulfovibrionaceae 60% Deltaproteobacteria - Desulfobulbaceae Deltaproteobacteria - Desulfobacteraceae Deltaproteobacteria - Desulfobacterales Deltaproteobacteria - Desulfarculaceae Betaproteobacteria - Hydrogenophilaceae Betaproteobacteria - Oxalobacteraceae 50% Betaproteobacteria - Comamonadaceae

% % reads Alphaproteobacteria - Rhodobacteraceae Proteobacteria Nitrospirae - OPB95 Nitrospirae - Nitrospirales Firmicutes - uncultured Clostridiales 40% Firmicutes - Syntrophomonadaceae Firmicutes - Ruminococcaceae Firmicutes - Peptococcaceae Firmicutes - JTB215 Firmicutes - Gracilibacteraceae Firmicutes - Family_XII_Incertae_Sedis 30% Firmicutes - Clostridiaceae Firmicutes - Clostridiales Elusimicrobia - Lineage_IV Deferribacteres - LCP-89 Cyanobacteria 20% Chloroflexi - vadinBA26 Chloroflexi - MSBL5 Chloroflexi - GIF9 Chloroflexi - Anaerolineaceae Chloroflexi - Anaerolineae CandidatedivisionOP9 10% Bacteroidetes - vadinHA17 - uncultured Bacteroidetes - vadinHA17 Bacteroidetes - VC2.1_Bac22 Bacteroidetes - WCHB1-69 Bacteroidetes - SB-5 Bacteroidetes - SB-1 0% Bacteroidetes - Flavobacteriaceae Env B4 S B1 B7 S B5 Env B1 3S B3 S B6 3S B7 S Env B3 S B5 B2 S B8 Bacteroidete - Cytophagaceae Bacteroidetes - Marinilabiaceae 25°C 10°C 25°C 10°C 25°C 10°C Bacteroidetes - BD2-2 SZ SMTZ MZ Actinobacteria - OPB41 Bacteria - Other

Figure S2. Relative abundance of the bacteria in all slurries and environmental samples at family level, normalized to 100%. Only those families that were present at an abundance >1% in at least one sample were included. SZ: Sulfate zone, SMTZ: Sulfate-methane transition zone; MZ: Methane zone, Env: Original Sediment Sample belonging to the indicated biogeochemical zone. S: 20mM sulfate, 3S:3mM sulfate is used as electron acceptor in slurries. Slurries that were not labeled with 'S' or '3S' were incubated without sulfate.

Bacteria OPB41 BD2-2 Bacteroidales Cytophagales Flavobacteriales SB-1 SB-5 Sphingobacteriales VC2.1Bac22 vadinHA17 vadinHA17uncultured Candidatedivision OP9 Anaerolineae Anaerolineales GIF9 MSBL5 vadinBA26 Chloroplast LCP89 LineageIV Clostridiales Nitrospirales Burkholderiales Hydrogenophilales Desulfarculales Desulfobacterales Desulfovibrionales Desulfuromonadales Sva0485 Campylobacterales Gammaproteobacteria Alteromonadales Pseudomonadales Thiotrichales LK-44f MSBL2 PBS-18 Spirochaetaceae Acholeplasmatales Temperature Sulfide Sulfate Methane Butyrate Acetate VFA Bacteria OPB41 Actinobacteria BD2-2 Bacteroidales Cytophagales Flavobacteriales SB-1 Bacteroidetes SB-5 Sphingobacteriales VC2.1 Bac22 vadinHA17 vadinHA17uncultured Candidate division OP9 Candidate division OP9 Anaerolineae Anaerolineales GIF9 Chloroflexi MSBL5 vadinBA26 Chloroplast Cyanobacteria LCP-89 Deferribacteres Lineage IV Elusimicrobia Clostridiales Firmicutes Nitrospirales Nitrospirae Burkholderiales Hydrogenophilales Desulfarculales Desulfobacterales Desulfovibrionales Desulfuromonadales Proteobacteria Sva0485 Campylobacterales Gammaproteobacteria Alteromonadales Pseudomonadales Thiotrichales LK-44f MSBL2 Spirochaetes PBS-18 Spirochaetaceae Acholeplasmatales Tenericutes Temperature Sulfide Sulfate Methane Butyrate Acetate VFA

-1 0 1

Figure S3. Heatmap representing the correlation between bacterial orders present at a relative abundance >1% of total reads across the 12 slurry samples analyzed and experimental parameters. Correlations were determined by means of the two tailed Spearman's Rank Order Correlation test. The heatmap colors represent the relative percentage of the microbial order assignments. Square colors shifting towards bright green indicate strong correlation. cetr n lris Sure ta wr nt aee wt '' r 3' ee nuae without incubated were sulfate. '3S' or 'S' with labeled not were that Slurries slurries. in acceptor electron as used is sulfate 3S:3mM sulfate, 20mM S: zone. biogeochemical indicated the zon Methane MZ: zone; transition methane Sulfate SMTZ: zone, Sulfate SZ: graph. the in included were sample one least at in abundance >1% an at present were that families those Only 100%. to normalized level, family S Figure

100% Marine Hydrothermal Vent Group (MHVG)

Hadesarchaea

90% Woesearchaeota (DHVEG-6) 4 . Thaumarchaeota - MCG Relative abundance of the the of abundance Relative 80% Thaumarchaeota - Group C3 70% Euryarchaeota - Unclassified Thermoplasmatales

Euryarchaeota - MBG-D and DHVEG-1 60% Euryarchaeota - 19c-33

50% Euryarchaeota - Methanosarcinaceae % % reads Euryarchaeota - Methanosaetaceae 40% Euryarchaeota - Unclassified Methanomicrobiales

30% Euryarchaeota - Methanomicrobiaceae Euryarchaeota - Methanomicrobiales - EJ-E01 A rchaea 20% Euryarchaeota - Methanomicrobiales - C19A

e. Env: e. Euryarchaeota -ANME-1 10%

Euryarchaeota - ANME-1b in a in 0% Euryarchaeota - Methanobacteriaceae ll slurries and environmental samples at samples environmental and slurries ll Original Env B4 S B1 3S B7 S B5 Env B1 3S B3 S B6 3S B7 S Env B3 S B5 B2 S B8 Euryarchaeota - Unclassified Halobacteriales 25°C 10°C 25°C 10°C 25°C 10°C Aenigmarchaeota - Deep Sea Euryarchaeotic Group(DSEG) SZ SMTZ MZ Bathyarchaeota

Sediment S ample belonging to belonging ample - familyassignments. Square colors shifted towardsbright greenindicate strong correlation. Rank Spearman's OrderCorrel tailed two the of means experimental by determined and were analyzed Correlations samples parameters. slurry 12 the across reads total of >1% abundance S Figure Unclassified Unclassified Unclassified Unclassified Unclassified Methanomicrobiaceae MBG-D and DHVEG-1 Methanosarcinaceae 5 Methanomicrobiales Thermoplasmatales AMOS1A-4113-D04 . Methanosaetaceae H ation test. The heatmapcolors represent the relative percentageof the microbial Halobacteriales eat Temperature map ANT06-05 Group Group C3 ANME-1b Methane Butyrate Acetate EJ-E01 MBG-B 19c-33 Sulfate Sulfide C19A showing MCG VFA

Unclassified Halobacteriales

ANME-1b the correlation between archaeal families present at a relative a at present familiesarchaeal betweencorrelation the C19A EJ-E01 Methanomicrobiaceae Unclassified Methanomicrobiales Methanosaetaceae Methanosarcinaceae 19c-33 AMOS1A-4113-D04 ANT06-05 MBG-D and DHVEG-1 Unclassified Thermoplasmatales Group C3 MBG-B MCG Temperature Sulfide -1 Sulfate Methane

0 Butyrate Acetate

1 VFA Thaumarchaeota Thermoplasmata Methanomicrobia Halobacteria

100%

90%

80% GlcA OL 70% TMO 60% PC

50% DMPE Betaine 40% MH

30% LPC IPL Relative AbundanceRelative(%) IPL PI 20% PG 10% PE DH 0% Env-SZ B4 B1 B7 B5 Env-SMZ B1 B3 B6 B7 Env-MZ B3 B5 B2 B8 PA S20 S0 S20 S0 S3 S20 S3 S20 S20 S0 S20 S0 25⁰C 10⁰C 25⁰C 10⁰C 25⁰C 10⁰C SZ SMTZ MZ Figure S6: Percent relative abundance of IPLs in all enrichment slurries and environmental samples used for molecular analysis. Env = Original Sediment Samples belonging to the indicated biogeochemical zone, S20 = 20 mM sulfate added, S0 = 0 mM sulfate added, S3 = 3 mM sulfate added, °C indicates incubation temperature, SZ = sulfate zone, SMTZ = sulfate-methane transition zone, MZ = methane zone.

IPL key: PA = phosphatidic acid, DH = dihexose, PE = phosphatidylethanolamine, PG = phosphatidylglycerol, PI = phosphatidylinositol, LPC = lyso-phosphatidylcholine, MH = monohexose, DMPE = dimethylphosphatidylethanolamine, PC = phosphatidylcholine, TMO = trimethylornithine, OL = ornithine, GlcA = glucuronic acid. 100% 90% TMO 80% PC 70% DMPE 60% Betaine 50% MH 40% LPC 30% PI 20% ND IPL RelativeIPL Abundance (%) 10% PG 0% PE B5 B6 B7 B8 B3 B4 B1 B2 DH S0 S0 S0 S0 S20 S20 S20 S20 PA 25⁰C 10⁰C 25⁰C 10⁰C

Figure S7: Percent relative abundance of IPLs in all methane zone enrichment slurries. IPL relative abundances for all methane zone slurries are combined to show full comparison between slurries. S20 = 20 mM sulfate added, S0 = 0 mM sulfate added, °C indicates incubation temperature, ND: Not Determined.

SMS10 PI 1.0 SS10 SME ME GlcA PG BBFPA SMs25 MH 0.5 MS25 BBC1 SMS25 OL BO BBVBBBBBS5 PE SMs10 BBM BAO

BCA2 0 0.0

PC BBWBBS

0.5 BBVH

CCA2 -

SZs25DH LPC

MZs10 1.0 - Ss10 SS25

BCG

1.5

- 2.0

- Ms25 1

DMPEBetaine - TMO

-3 -2 -1 0 1 2 3

CCA1

Figure S8: Canonical correspondence analysis of intact polar lipid (IPL) distribution, bacterial taxa distribution, and growth conditions for all enrichment slurries.

Black text, culture condition key: S = sulfate zone, SM = sulfate-methane transition zone, M = methane zone, S = high sulfate amendment, s = low sulfate amendment, 25 = 25°C, 10 = 10°C.

Green text, IPL key: MH = monohexose, PA = phosphatidic acid, DH = dihexose, DMPE = dimethylphosphatidylethanolamine, PE = phosphatidylethanolamine, PG = phosphatidylglycerol, OL = ornithine, GlcA =glucuronic acid, PC = phosphatidylcholine, TMO = trimethylornithine, MMPE = monomethylphosphatidylethanolamine, PI = phosphatidylinositol, LPC = lyso-phosphatidylcholine.

Blue text, bacterial taxa key: BAO = Bacteria - Actinobacteria - OPB41; BBB = Bacteria - Bacteroidetes - BD2-2; BBC1 = Bacteria - Bacteroidetes - Cytophagaceae; BBF = Bacteria - Bacteroidetes - Flavobacteriaceae; BBM = Bacteria - Bacteroidetes - Marinilabiaceae; BBS = Bacteria - Bacteroidetes - SB-1; BBS5 = Bacteria - Bacteroidetes - SB-5; BBV = Bacteria - Bacteroidetes - VC2.1_Bac22; BBVH = Bacteria - Bacteroidetes - vadinHA17; BBW = Bacteria - Bacteroidetes - WCHB1-69; BCA2 = Bacteria - Chloroflexi - Anaerolineaceae; BCG = Bacteria - Chloroflexi - GIF9; BCO = Bacteria - Candidate division OP9; BO = Bacteria other.