Free energy yield for methagenomic reactions. kJ/mol reaction. kJ/L fluid. Limiting Reactant. Hydrogenotrophic methanogenesis (4H2 + CO2 -> CH4 + 2H2O).
Supplementary Material Table S1. Geochemical and isotopic data for samples collected from Santa Elena Ophiolite (Average data presented in Table 1)1 DOC (µM) 7.9 3.5 5.4 101
TDN (µM) bdl 0.3 0.4 6.6
Geochemical Parameters NOx NO2 NH4+ DON (µM) (µM) (µM) (µM) 0.3 0.03 1.57 bdl 0.2 0.01 1.3 bdl 1.8 0.01 1.16 bdl 2.7 0.07 0.69 3.2
Sample Name Q.Danta Q.Danta Q.Danta Q.Danta Upstream Q.Danta 105 7 2.5 0.07 Upstream Q.Danta 103 6.1 2.4 0.07 Upstream Spring 8 73.1 14.8 0.3 0.01 Spring 8 70.4 13.4 0.3 0.01 Spring 8 76 14.5 0.3 0.03 Spring 9 45.3 2.4 0.4 0.01 Spring 9 36.8 0.9 0.2 0.03 Spring 9 41.4 1.9 0.1 0.03 Murciélago 67.3 1.8 0.4 0.05 Upstream Murciélago 67.6 1.9 0.3 0.07 Upstream Murciélago 66.3 1.5 0.3 0.05 Upstream R.Calera 54.5 2.6 1.7 0.03 R.Calera 63.7 2.6 1.9 0.03 R.Calera 60.3 2.6 1.9 0.05 P. Murciélago 8.2 61.3 58.5 0.01 P. Murciélago 12.1 62.2 57.8 0.03 P. Murciélago 14.6 63.7 58.7 0.03 P. Nuevo 12.6 3.6 0.2 0.03 P. Nuevo 19.6 3.6 0.3 0.01 P. Nuevo 28.4 3.4 0.1 0.03 P. Aguas 26 38.3 36.7 0.03 Calientes P. Aguas 28.3 40.4 37.2 0.03 Calientes P. Aguas 23.3 38.9 36.6 0.03 Calientes SD: standard deviation; bdl: below detection limit
PO43(µM) 0.09 0.05 0.09 1.52
TDP (µM) 0.17 0.08 0.08 0.17
CH4 (µM) 145 145 145 0.36
Gaseous Geochemistry and isotopic composition H2 DIC δ 13C-CH4 δ 13C-CH4 δ 13C-CO2 2 (µM) (µM) (‰) SD (‰) (‰) -43.86 0.22 45.62 119 -18.66 -44.07 0.19 15.53 85 -22.68 -44.18 0.18 53.6 175 -18.85 -36.4 18.8 8.33 632 -17.04
δ 13C-CO2 SD (‰) 0.81 1.13 0.65 0.65
0.48
4
1.56
0.27
0.29
-37.67
18.35
18.22
608
-17.86
0.57
0.55
3.2
1.6
0.13
0.33
-37.97
16.02
0.95
552
-18.78
0.77
1.77 1.77 1.64 0.75 0.82 0.96 0.14
12.7 11.3 12.5 1.3 bdl 0.9 1.3
0.22 0.22 0.22 0.31 0.22 0.27 1.6
0.13 0.13 0.13 0.03 0.08 0.08 0.08
905 889 818 944 903 890 14
-1.71 -1.54 0.51 -2.51 -1.86 -2.22 1.77
0.3 0.36 0.31 0.48 0.45 0.24 0.63
4.27 6.48 21.8 36.19 34.8 88.35 0.8
222 315 144 293 237 233 506
-17.99 -15.94 -19.79 -17.35 -22.72 -22.35 -19.98
0.89 0.44 1.04 0.66 1.91 2.25 0.68
0.14
1.4
1.52
0.41
15
1.2
0.65
0.69
529
-20.08
0.76
0.14
1.1
1.56
0.13
14
0.98
0.62
0.78
566
-19.43
0.66
0.07 0.14 0.07 2.11 1.84 2.38 4.08 3.95 4.36 0.89
0.8 0.6 0.6 0.7 2.6 2.6 bdl bdl bdl 0.7
1.9 1.77 1.9 2.42 2.85 2.68 1.6 1.64 1.82 2.55
0.22 0.13 0.13 0.41 0.41 0.41 0.55 0.74 0.55 1.06
0.42 0.22 0.13 0.32 0.24 0.2 0.11 0.4 0.43 0.27
-22 -22.83 -9.67 3.07 5.59 9.44 0.71 -22.13 -29.57 -11.51
19.23 23.22 35.32 40.7 52.66 66.39 57.07 20.35 14.85 22.79
69.22 0.39 1.36 0.62 1.05 1.21 0.65 1.08 0.65 1.78
720 635 635 723 792 729 437 406 410 632
-18.76 -18.69 -18.46 -19.63 -19.51 -19.24 -18.18 -18.72 -18.84 -18.89
0.5 0.61 0.63 0.48 0.48 0.59 0.84 0.73 0.65 0.65
1.64
1.5
2.77
1.11
0.17
2.52
35.59
0.68
687
-18.59
0.52
1.84
0.5
2.89
1.11
0.14
16.91
40.67
0.39
614
-18.68
0.57
1
Analyses were done as contracted services at the Joye Biogeochemistry Laboratory (University of Georgia, GA, USA; http://www.joyeresearchgroup.uga.edu/). The analytical methods used were: combustion for DOC and TDN, chemiluminescence for NOx, spectrophotometry for NO2, NH4+, PO43- and TDP, gas chromatography for H2, and cavity ringdown spectrometry for CH4, DIC and their respective isotopes. DON was calculated from TDN and DIN values. 2
Samples with methane concentration below 0.3 µM were within the limit of the detection and resulted in δ13C-CH4 signature with an elevated standard deviation (16-66 ‰)
Table S2. Quantification of methane-cycling genes searched for within the metagenomes (normalized reads per fragment). Data was normalized to the total number of fragments in the smaller metagenome. KEGG ID’s denoted by two asterisks (**) represents genes that were found in both metagenomes, while those denoted by one asterisk (*)were found only in the Spring 9 metagenome. No genes were found only in the Murcielago Upstream metagenome.
KEGG ID K00123** K00124** K00125* K00126 K00127** K00200 K00201 K00202 K00203 K00204 K00205* K11260* K11261 K00672* K01499* K00319* K13942 K00440** K00441** K00442 K00443 K00320* K00577* K00578* K00579* K00580* K00581* K00582
Gene formate dehydrogenase formate dehydrogenase formate dehydrogenase formate dehydrogenase formate dehydrogenase formylmethanofuran dehydrogenase formylmethanofuran dehydrogenase formylmethanofuran dehydrogenase formylmethanofuran dehydrogenase 4Fe-4S ferredoxin 4Fe-4S ferredoxin 4Fe-4S ferredoxin formylmethanofuran dehydrogenase formylmethanofuran--tetrahydromethanopterin N-formyltransferase methenyltetrahydromethanopterin cyclohydrolase methylenetetrahydromethanopterin dehydrogenase 5,10-methenyltetrahydromethanopterin hydrogenase coenzyme F420 hydrogenase coenzyme F420 hydrogenase coenzyme F420 hydrogenase coenzyme F420 hydrogenase 5,10-methylenetetrahydromethanopterin reductase tetrahydromethanopterin S-methyltransferase tetrahydromethanopterin S-methyltransferase tetrahydromethanopterin S-methyltransferase tetrahydromethanopterin S-methyltransferase tetrahydromethanopterin S-methyltransferase tetrahydromethanopterin S-methyltransferase
Name fdoG, fdfH fdoH E1.2.1.2B2 fdsD fdoI fwdA, fmdA fwdB, fmdB fwdC, fmdC fwdD, fmdD fwdH fwdF, fmdF fwdG fwdE, fmdE ftr mch mtd hmd frhA frhB frhD frhG mer mtrA mtrB mtrC mtrD mtrE mtrF
Normalized Reads per Fragment (RPF) Spring 9 Murcielago Upstream 1792.91 9445.79 1030.46 3903.61 175.33 0.00 0.00 0.00 1145.03 5580.30 8.62 0.00 150.79 0.00 0.00 0.00 131.78 0.00 0.00 0.00 152.09 0.00 165.88 0.00 0.00 0.00 18.40 0.00 139.34 0.00 129.80 0.00 0.00 0.00 0.00 0.00 364.19 24.21 0.00 0.00 0.00 0.00 178.31 0.00 121.23 0.00 103.07 0.00 114.32 0.00 125.78 0.00 148.24 0.00 0.00 0.00
K00583* K00584* K00399* K00401* K00402* K03388* K03389* K03390* K00925** K00625** K13788 K01895** K00192* K00193* K00194* K00195* K00196 K00197* K00198
K14082 K14083 K14084 K16176 K16177 K16178 K16179 K16157 K16158 K16159 K16161
tetrahydromethanopterin S-methyltransferase tetrahydromethanopterin S-methyltransferase methyl-coenzyme M reductase methyl-coenzyme M reductase methyl-coenzyme M reductase heterodisulfide reductase heterodisulfide reductase heterodisulfide reductase acetate kinase phosphate acetyltransferase phosphate acetyltransferase AMP-forming acetyl-CoA synthetase acetyl-CoA decarbonylase/synthase acetyl-CoA decarbonylase/synthase acetyl-CoA decarbonylase/synthase acetyl-CoA decarbonylase/synthase carbon-monoxide dehydrogenase acetyl-CoA decarbonylase/synthase carbon-monoxide dehydrogenase [methyl-Co(III) methanol-specific corrinoid protein]:coenzyme M methyltransferase methanol---5-hydroxybenzimidazolylcobamide Co-methyltransferase methanol corrinoid protein [methyl-Co(III) methylamine-specific corrinoid protein]:coenzyme M methyltransferase trimethylamine---corrinoid protein Co-methyltransferase trimethylamine corrinoid protein methylamine---corrinoid protein Co-methyltransferase monomethylamine corrinoid protein dimethylamine---corrinoid protein Co-methyltransferase dimethylamine corrinoid protein methane mooxygenase methane mooxygenase methane mooxygenase methane mooxygenase
K10944
methane/ammonia monooxygenase
K14080 K04480 K14081
mtrG mtrH mcrA mcrB mcrG hdrA hdrB hdrC ackA pta pta acs cdhA cdhC cdhD, acsD cdhB cooF cdhE, acsC cooS, acsA mtaA mtaB mtaC mtbA mttB mttC mtmB mtmC mtbB mtbC mmoX mmoY mmoZ mmoC pmoA, amoA
223.51 153.48 152.15 154.33 46.90 284.39 11.06 174.66 569.43 3372.74 0.00 4706.60 140.33 19.96 121.80 337.54 0.00 173.17 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 222.76 452.66 0.00 9737.09 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00
0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
K00368 K15864 K14028
nitrite reductase (NO-forming) nitrite reductase (NO-forming) / hydroxylamine reductase methanol dehydrogenase
nirK nirS mdh1, mxaF
0.00 0.00 0.00
0.00 0.00 0.00
Table S3. Free energy yield for methagenomic reactions kJ/mol reaction kJ/L fluid Hydrogenotrophic methanogenesis (4H2 + CO2 -> CH4 + 2H2O) Q.Danta -54.41 -5.20 x 10-4 Spring 9 -56.53 -7.51 x 10-4 Spring 8 -40.91 -1.11 x 10-4 Murcielago Upstream -48.04 -9.05 x 10-6 Q.Danta Upstream -85.60 -1.96 x 10-4 R.Calera -95.04 -5.62 x 10-4 P. Murcielago -69.55 -1.67 x 10-5 P. Nuevo -61.48 -1.22 x 10-5 P. Aguas Calientes -70.70 -1.67 x 10-5 Acetoclastic methanogenesis (CH3COO- + H+ -> CH4 + CO2); Acetate = 10% of DOC Q.Danta -31.65 -1.77 x 10-4 Spring 9 -28.90 -1.19 x 10-3 Spring 8 -30.44 -2.23 x 10-3 Murcielago Upstream -32.07 -2.15 x 10-3 Q.Danta Upstream -41.82 -4.31 x 10-3 R.Calera -40.56 -2.41 x 10-3 P. Murcielago -36.35 -4.23 x 10-4 P. Nuevo -38.60 -7.80 x 10-4 P. Aguas Calientes -40.06 -1.04 x 10-3 + Acetoclastic methanogenesis (CH3COO + H -> CH4 + CO2); Acetate = 1% of DOC Q.Danta -25.95 -1.45 x 10-4 Spring 9 -23.19 -9.55 x 10-4 Spring 8 -24.73 -1.81 x 10-3 Murcielago Upstream -26.37 -1.77 x 10-3 Q.Danta Upstream -36.12 -3.72 x 10-3 R.Calera -34.85 -2.07 x 10-3 P. Murcielago -30.64 -3.57 x 10-4 P. Nuevo -32.89 -6.65 x 10-4 P. Aguas Calientes -34.35 -8.88 x 10-4
Limiting Reactant hydrogen hydrogen hydrogen hydrogen hydrogen hydrogen hydrogen hydrogen hydrogen acetate acetate acetate acetate acetate acetate acetate acetate acetate acetate acetate acetate acetate acetate acetate acetate acetate acetate
Table S4. Free energy yield for methanotrophic reactions kJ/mol reaction kJ/L fluid -2 + Anaerobic Methane Oxidation with Sulfate (CH4 + SO4 + H -> CO2 + HS + 2 H2O) Q.Danta -47.64 -1.64 x 10-4 Spring 9 -49.06 -1.69 x 10-4 Spring 8 -48.95 -1.68 x 10-4 Murcielago Upstream -33.65 -4.82 x 10-4 Q.Danta Upstream -24.50 -7.98 x 10-6 R. Calera -22.53 -5.75 x 10-6 P. Murcielago -19.08 -4.86 x 10-6 P. Nuevo -20.94 -6.50 x 10-6 P. Aguas Calientes -25.24 -4.84 x 10-6 Anaerobic Methane Oxidation with Nitrate (CH4 + 4NO3 -> CO2 + 4NO2 + 2H2O) Q.Danta -585.49 -1.09 x 10-4 Spring 9 -570.96 -2.57 x 10-5 Spring 8 -577.77 -4.28 x 10-5 Murcielago Upstream -534.22 -3.69 x 10-5 Q.Danta Upstream -539.51 -1.76 x 10-4 R. Calera -542.04 -1.38 x 10-4 P. Murcielago -574.05 -1.46 x 10-4 P. Nuevo -525.20 -1.63 x 10-4 P. Aguas Calientes -566.07 -1.08 x 10-4 Aerobic Methanotrophy (CH4 + 2O2 -> CO2 + 2H2O); O2= 0.1% saturation Q.Danta -818.09 -4.09 x 10-5 Spring 9 -819.22 -4.10 x 10-5 Spring 8 -819.10 -4.10 x 10-5 Murcielago Upstream -785.53 -3.93 x 10-5 Q.Danta Upstream -772.73 -3.86 x 10-5 R. Calera -772.69 -3.86 x 10-5 P. Murcielago -766.42 -3.83 x 10-5 P. Nuevo -772.61 -3.86 x 10-5 P. Aguas Calientes -765.14 -3.83 x 10-5 Aerobic Methanotrophy (CH4 + 2O2 -> CO2 + 2H2O); O2= 1.0% saturation Q.Danta -829.50 -4.15 x 10-4 Spring 9 -830.63 -4.15 x 10-4
Limiting Reactant sulfate sulfate sulfate methane methane methane methane methane methane nitrate nitrate nitrate nitrate methane methane methane methane methane oxygen oxygen oxygen oxygen oxygen oxygen oxygen oxygen oxygen oxygen oxygen
Spring 8 Murcielago Upstream Q.Danta Upstream R. Calera P. Murcielago P. Nuevo P. Aguas Calientes
-830.51 -796.94 -784.14 -784.11 -777.83 -784.02 -776.55
-4.15 x 10-4 -3.98 x 10-4 -2.52 x 10-4 -1.97 x 10-4 -1.95 x 10-4 -2.40 x 10-4 -1.47 x 10-4
oxygen oxygen methane methane methane methane methane
