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transcript
Supplementary Material for
Methanogenic biodegradation of iso-alkanes and cycloalkanes during long-term incubation with oil sands tailings
Tariq Siddique†,*, Kathleen Semple‡, Carmen Li⁋, and Julia M. Foght‡,
†Department of Renewable Resources, University of Alberta, Edmonton, AB T6G 2E3, Canada‡Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada⁋Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada
Corresponding author e-mail: tariq.siddique@ualberta.ca
The Supplementary Material contains three figures (Figure S1-S3) and eight tables (Table S1-S8).
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Table S1: Experimental set-up to study biodegradation of iso-alkanes and cycloalkanes in MFT and aqueous medium.
Culture designation
Matrix MFT depth
Additional inoculum a
Volumes (mL) b
Treatment types c
MFT-6m MFT slurry
6 m None MFT: 50 Medium: 50
Unamended Amended Sterile
amendedMFT-6m-N MFT
slurry6 m Naphtha
diluent enrichment culture
MFT: 50 Medium: 40Culture: 10
Unamended Amended Sterile
amendedMFT-6m-P MFT
slurry6 m Paraffinic
diluent enrichment culture
MFT: 40 Medium: 40Culture: 20
Unamended Amended Sterile
amendedMFT-31m MFT
slurry31 m None MFT: 50
Medium: 50 Unamended Amended Sterile
amendedNo-MFT-P Aqueous
phasen/a d Paraffinic
enrichment culture
Medium: 80Culture: 20
Amended
a MFT-derived primary naphtha enrichment culture grown on naphtha for ~270 d or MFT-derived primary paraffinic diluent enrichment culture grown for ~500 d on paraffinic diluent (see Methods)b Each methanogenic microcosm (158mL volume) contained 100 mL total of experimental material with headspace of 30% CO2, balance N2.c Treatments were: Unamended, no hydrocarbon mixture added; Amended, mixture of eight hydrocarbons added to a total concentration of 200 mg L-1 experimental material (see Methods); and Sterile amended, heat-killed MFT subsequently amended with hydrocarbon mixture.d not applicable; no MFT added.
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Table S2: Measured and theoretical maximum CH4 production by alkane-amended cultures calculated from biodegradation measurements (Figures 1 and S1).
Hydro-carbon
Mass of hydro-carbon added (mmol) a
Maximum CH4 expected (mmol)
% of hydro-carbon degraded b
Theoretical yield ofCH4 (mmol)
Total CH4
expected (mmol)
Total CH4
observed(mmol) c
% of expected CH4
MFT-6m 3-MC6 0.20 1.10 98 1.08 4.3 2.6 602-MC7 0.17 1.25 64 0.804-MC7 0.17 1.25 89 1.112-MC8 0.16 1.40 96 1.34MFT-6m-N 3-MC6 0.20 1.10 96.0 1.05 4.8 2.7 562-MC7 0.17 1.25 98.2 1.224-MC7 0.17 1.25 100.0 1.252-MC8 0.16 1.40 91.0 1.274MFT-6m-P 3-MC6 0.20 1.10 96.0 1.05 4.8 3.0 622-MC7 0.17 1.25 95.0 1.184-MC7 0.17 1.25 99.5 1.242-MC8 0.16 1.40 99.2 1.38MFT-31m 3-MC6 0.20 1.10 98.5 1.08 4.9 3.6 732-MC7 0.17 1.25 98.5 1.234-MC7 0.17 1.25 99.5 1.242-MC8 0.16 1.40 100.0 1.40No-MFT-P 3-MC6 0.20 1.10 87 0.96 2.8 1.19 432-MC7 0.17 1.25 43 0.544-MC7 0.17 1.25 59 0.742-MC8 0.16 1.40 44 0.62
a Calculated from volumes of hydrocarbons used to prepare iso- and cycloalkane mixtureb Calculated from data shown in Figures 1 and S1.c The mass of methane produced from endogenous carbon sources in unamended (baseline) cultures was subtracted from the mass produced by amended cultures to calculate ‘Observed CH4’; see Methods.
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Table S3. Relative abundance of 16S rRNA gene pyrosequences affiliated with Bacteria in unamended and amended MFT-6m cultures
BacteriaUnamended Amended
Day120 815 120 815
Actinobacteria Coriobacteriales; Coriobacteriaceae 0.7 2.2 1.6 0.1Coriobacteriales; Coriobacteriaceae; Eggerthella 1.7 1.5 1.4 0.5Bacteroidetes Sphingobacteriales; Chitinophagaceae; Sediminibacterium 0.0 1.2 0.1 0.0Sphingobacteriales; vadinHA17 3.7 2.2 3.1 0.7ChloroflexiChloroflexi 0.2 1.2 0.9 1.1Anaerolineales; Anaerolineaceae 1.2 1.2 0.6 0.5Anaerolineales; Anaerolineaceae; Leptolinea 4.5 4.6 3.7 1.7Anaerolineales; Anaerolineaceae; uncultured 8.8 12.9 7.3 5.8FirmicutesFirmicutes 0.6 0.3 1.8 0.1Clostridiales; Family XI Incertae Sedis 1.4 0.0 1.3 0.2Clostridiales; Family XIII Incertae Sedis; Anaerovorax 1.2 0.3 0.6 0.0Clostridiales; Peptococcaceae; Desulfotomaculum 2.3 17.8 2.9 60.2Clostridiales; Ruminococcaceae; Fastidiosipila 1.6 0.0 2.0 0.2ProteobacteriaBurkholderiales; Comamonadaceae; Acidovorax 15.3 0.9 17.0 1.1Rhodocyclales; Rhodocyclaceae; uncultured 1.9 0.9 1.5 0.4Desulfobacterales; Desulfobacteraceae; Desulfobacterium 3.8 7.1 3.8 2.6Desulfobacterales; Desulfobulbaceae; Desulfocapsa 5.7 5.2 4.8 1.7Desulfuromonadales; Desulfuromonadaceae; Desulfuromonas 2.2 2.5 1.9 0.8Syntrophobacterales; Syntrophaceae; Smithella 10.5 7.7 9.7 11.2Syntrophobacterales; Syntrophaceae; Syntrophus 1.7 12.3 1.3 3.2Syntrophobacterales; Syntrophaceae; uncultured 0.7 2.2 0.9 1.5Gammaproteobacteria 6.6 3.1 3.9 0.4SpirochaetesSpirochaetales; Spirochaetaceae; uncultured 2.0 1.5 3.6 0.5SynergistetesSynergistales; Synergistaceae; Thermanaerovibrio 0.7 1.2 0.4 0.8Other Bacteria (>1%)Candidate_division_OP11 2.2 0.6 1.0 0.0Candidate_division_WS6 2.7 2.5 3.3 0.1Other Bacteria (<1%) 16.2 6.8 19.4 4.6
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≥ 61%0%
Table S4. Relative abundance of 16S rRNA gene pyrosequences affiliated with Bacteria in unamended and amended MFT-31m cultures
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≥ 47%0% ≤
BacteriaUnamended Amended
Day182 420 815 182 420 815
Actinobacteria Microbacteriaceae; Microbacterium 0.8 0.9 2.1 1.1 0.6 0.3Coriobacteriaceae 0.7 0.4 0.9 1.3 0.6 0.1BacteroidetesChitinophagaceae; Sediminibacterium 0.7 0.3 1.8 0.5 0.3 0.3ChloroflexiAnaerolineaceae; Leptolinea 1.8 1.5 3.3 2.2 0.9 1.1
Anaerolineaceae; uncultured 5.7 6.712.2 6.1 3.7 4.5
Firmicutes Clostridia 2.1 0.3 0.0 2.4 0.1 0.1Peptococcaceae; Desulfotomaculum 0.8 2.1 1.2 0.7 46.8 45.5ProteobacteriaComamonadaceae 1.4 1.1 0.9 1.2 0.6 0.4Comamonadaceae; Acidovorax 31.4 23.2 3.9 30.7 12.2 0.5Comamonadaceae; Brachymonas 1.7 1.8 0.8 2.3 1.1 0.2
Hydrogenophilaceae; Thiobacillus 10.9 13.021.5 10.0 5.5 3.3
Rhodocyclaceae; uncultured 2.0 3.8 3.4 2.6 3.1 0.8Oxalobacteraceae; Herbaspirillum 1.7 0.5 0.1 1.3 0.1 0.0Desulfobacteraceae; Desulfobacterium 0.4 0.7 1.0 0.5 0.8 1.3Desulfobulbaceae; Desulfobulbus 0.7 1.1 0.9 0.1 0.1 0.0Desulfobulbaceae; Desulfocapsa 0.8 1.1 1.4 1.3 0.6 0.4Desulfobulbaceae; Desulfurivibrio 4.8 7.3 4.8 2.0 1.6 1.0Geobacteraceae; Geobacter 0.5 0.7 1.2 0.3 0.7 0.0Syntrophaceae; Smithella 1.1 1.7 2.5 1.6 1.7 28.4Syntrophaceae; Syntrophus 0.5 1.2 1.5 0.5 0.5 2.6Alteromonadaceae; uncultured 1.3 1.6 1.8 1.2 1.0 0.5Chromatiales; Chromatiaceae 1.1 2.9 7.1 1.6 2.4 1.8Pseudomonadaceae; Pseudomonas 2.0 0.4 0.5 1.5 0.2 0.0Xanthomonadaceae 2.0 0.8 0.1 1.1 0.6 0.0Other Bacteria (>1%) Candidate_division_WS6 0.9 0.2 0.6 1.3 0.3 0.2Nitrospiraceae; uncultured 0.2 1.2 0.8 0.2 0.0 0.0
Other Bacteria (<1%) 21.9 23.823.7 24.3 13.7 6.7
Table S5. Relative abundance of 16S rRNA gene pyrosequences affiliated with Bacteria in unamended and amended inoculated MFT-6m-P cultures and in amended inoculated medium (No-MFT-P).
Bacteria
MFT-6m-PUnamended
No-MFT-P Amended
MFT-6m-P Amended
Day420 815 420 815 420 815
ActinobacteriaCoriobacteriaceae 0.0 1.1 0.1 0.5 0.5 0.8Coriobacteriaceae; Eggerthella 0.0 0.6 1.0 1.0 0.8 0.4BacteroidetesChitinophagaceae; Sediminibacterium 0.0 0.2 0.6 0.5 0.0 0.0Sphingobacteriales; vadinHA17 2.3 2.0 0.4 2.7 0.5 0.3ChloroflexiChloroflexi 1.2 1.2 0.0 1.9 1.9 1.6Anaerolineaceae 0.0 1.4 0.1 0.7 1.1 0.5Anaerolineaceae; Leptolinea 4.7 4.4 0.8 2.4 1.9 1.2Anaerolineaceae; uncultured 5.8 10.4 3.6 6.4 3.2 4.0Firmicutes Clostridiaceae; Clostridium 1.2 0.0 0.1 0.0 0.0 0.1Family XI Incertae Sedis 1.2 0.2 0.4 0.3 0.0 0.0Family XIII Incertae Sedis; Anaerovorax 1.2 0.0 0.0 0.0 0.3 0.0Peptococcaceae; Desulfotomaculum 30.2 24.3 43.9 44.6 56.8 25.1Ruminococcaceae; Fastidiosipila 1.2 0.2 0.3 0.0 1.1 0.0Proteobacteria Comamonadaceae 1.2 0.0 0.0 0.0 0.0 0.3Comamonadaceae; Acidovorax 15.1 3.7 0.4 0.1 8.3 0.8Comamonadaceae; Rubrivivax 1.2 0.0 0.0 0.0 0.5 0.0Rhodocyclaceae; uncultured 1.2 0.2 0.0 0.0 0.5 0.2Desulfobacteraceae; Desulfobacterium 2.3 2.6 3.0 3.7 1.1 1.3Desulfobulbaceae; Desulfocapsa 3.5 5.6 7.7 7.3 1.3 2.7Desulfovibrionaceae; Desulfovibrio 0.0 0.0 18.5 0.5 0.0 0.0Desulfuromonadaceae; Desulfuromonas 0.0 1.1 0.0 0.0 0.3 0.1Geobacteraceae; Geobacter 0.0 0.6 0.3 1.8 0.5 0.0Syntrophaceae 0.0 0.5 0.3 0.0 0.0 0.8Syntrophaceae; Smithella 3.5 13.5 2.3 7.3 3.7 48.4Syntrophaceae; Syntrophus 9.3 14.8 2.7 10.7 1.6 7.0Gammaproteobacteria 3.5 1.2 0.0 0.0 2.1 0.2Spirochaetes Spirochaetaceae; uncultured 2.3 0.8 0.6 0.0 0.5 0.3Synergistetes Synergistaceae; Thermanaerovibrio 4.7 0.9 4.4 2.7 0.8 0.2Other Bacteria (>1%) Bacteria 2.3 0.9 0.1 0.0 0.3 0.7
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Candidate_division_OP11 0.0 0.0 0.0 0.0 1.1 0.0Acidobacteriaceae; uncultured 1.2 0.0 0.0 0.0 0.3 0.0Other Bacteria (<1%) 0.0 7.5 8.3 4.9 8.9 3.0
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≥ 57%0% ≤
Table S6. Relative abundance of 16S rRNA gene pyrosequences affiliated with Archaea in unamended and amended MFT-6m
ArchaeaUnamended Amended
Day120 815 120 815
Methanomicrobia Methanomicrobiales; Methanoregula 60.4 44.7 62.5 55.0Methanomicrobiales; Methanomicrobiaceae; Methanoculleus 1.3 9.8 2.4 11.2
Methanosarcinales; Methanosaetaceae; Methanosaeta 35.2 44.0 31.7 32.3
ThermoplasmataWCHA1-57 1.1 0.4 1.0 0.1Other Archaea (<1%) 1.9 1.3 2.4 1.4
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≥ 63%0% ≤
Table S7. Relative abundance of 16S rRNA gene pyrosequences affiliated with Archaea in unamended and amended MFT-31m
ArchaeaUnamended Amended
Day182 420 815 182 420 815
MethanobacteriaMethanobacteriales; Methanobacteriaceae; Methanobacterium 1.5 0.7 0.8 1.4 0.9 0.0Methanomicrobia Methanomicrobiales; Methanoregula 40.4 39.1 41.6 38.1 25.8 9.3Methanomicrobiales; Methanomicrobiaceae; Methanoculleus 2.8 6.0 7.2 4.4 15.4 27.3Methanosarcinales; Methanosaetaceae; Methanosaeta 43.6 44.5 42.4 47.3 51.4 61.9Methanosarcinales; Methanosarcinaceae; Methanomethylovorans 5.3 4.3 3.8 3.6 2.6 0.0Thermoplasmata Thermoplasmatales; Terrestrial Miscellaneous Gp (TMEG) 1.1 0.7 1.4 0.8 1.0 0.0WCHA1-57 2.8 2.7 1.7 2.9 1.5 0.0Other Archaea (<1%) 2.4 2.0 1.2 1.4 1.5 1.4
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≥ 62%0% ≤
Table S8. Relative abundance of 16S rRNA gene pyrosequences affiliated with Archaea in unamended and amended inoculated MFT-6m-P cultures and in amended inoculum (no-MFT-P)
Archaea
MFT-6m-PUnamended
No-MFT-P Amended
MFT-6m-P Amended
Day420 815 420 815 420 815
Methanomicrobia Methanomicrobiales; Methanoregula 67.5 63.5 18.5 5.9 64.3 63.5Methanomicrobiales; Methanomicrobiaceae; Methanoculleus 2.3 4.3 36.1 52.5 3.5 5.3Methanomicrobiales; Methanomicrobiaceae 0.2 0.1 1.3 1.5 0.3 0.3Methanosarcinales; Methanosaetaceae; Methanosaeta 27.2 30.1 43.0 38.8 29.8 29.9Other Archaea (<1%) 2.9 2.0 1.1 1.3 2.0 1.1
10
≥ 68%0% ≤
Fig. S1. Cumulative CH4 production by alkane-amended or unamended MFT-6m inoculated with (A) a paraffinic hydrocarbon-degrading enrichment culture (MFT-6m-P) or (B) a naphtha-degrading enrichment culture (MFT-6m-N). Cumulative CH4 production from the paraffin-
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0 400 800 1200 16000
1
2
3
4
5
6
7
CH
4 (m
mol
)
Day
Unamended MFT Amended MFT Amended medium
0 400 800 1200 16000
20
40
60
80
100
120
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160
180
Res
idua
l hyd
roca
rbon
(%)
Day
0 400 800 1200 16000
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Res
idua
l hyd
roca
rbon
(%)
Day
0 400 800 1200 16000
1
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CH
4 (m
mol
)Day
Unamended MFT Amended MFT
0 400 800 1200 16000
20
40
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140R
esid
ual h
ydro
carb
on (%
)
Day
3-MC6
3-EC6
2-MC7
4-MC7
2-MC8
EcyC5
EcyC6
McyC6
n-C7
A
C
E
B
D
degrading inoculum without added MFT (No-MFT-P) is also shown in panel (A). Hydrocarbon biodegradation was calculated by reference to parallel sterile MFT controls and expressed as % residual hydrocarbon in the n-alkane-amended (C) MFT-6m-P or (D) MFT-6m-N cultures, or in (E) the amended paraffin-degrading culture with no MFT added (No-MFT-P). Data points are mean values (n=3) and error bars, where visible, represent the standard deviation. The abbreviations are: 3-MC6 (3-methylhexane), 3-EC6 (3-ethylhexane), 2-MC7 (2-methylheptane), 4-MC7 (4-methylheptane), 2-MC8 (2-methyloctane), EcyC5 (ethylcyclopentane), McyC6 (methylcyclohexane), EcyC6 (ethylcyclohexane) and nC7 (n-heptane).
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120 d 815 d
UnamendedMFT-6m
AmendedMFT-6m
UnamendedMFT-31m
AmendedMFT-31m
182 d 420 d 815 d
420 d 815 d
UnamendedMFT-6m-P
AmendedMFT-6m-P
AmendedNo-MFT-P
Archaeal reads Bacterial reads
Fig S2: Proportions of archaeal and bacterial reads from pyrosequencing of 16S rRNA genes.
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420 d 815 d
Peptococcaceae Other Firmicutes Proteobacteria Actinobacteria Chloroflexi Bacteroidetes Spirochaetes Synergistetes Other Bacteria
Methanoregula Methanomicrobiaceae Other Methanomicrobiales Methanosaetaceae Methanosarcinaceae Other Methanosarcinales Methanobacteriaceae Other Archaea
UnamendedMFT-6m-P
AmendedMFT-6m-P
UnamendedMFT-6m-P
AmendedMFT-6m-P
A
B
AmendedNo-MFT-P
AmendedNo-MFT-P
Fig. S3. Microbial community structure determined by 16S rRNA gene pyrosequencing of amended MFT-6m inoculated with paraffin-degrading enrichment culture (MFT-6m-P) and amended medium without MFT (No-MFT-P) during microbial metabolism of iso-alkanes and cycloalkanes to CH4. Graphs show proportions of (A) bacterial taxa and (B) archaeal taxa at 420 and 815 d incubation. “Other Bacteria” and “Other Archaea” are the sums of taxa individually present at ≤1% abundance.
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