Supplement Information

Effect of Naphtha Diluent on Greenhouse Gases and

Reduced Sulfur Compounds Emissions from Oil

Sands Tailings

Kathleen F. Gee, Ho Yin Poon, Zaher Hashisho and Ania C. Ulrich*

Address correspondence to Ania C. Ulrich, Department of Civil and Environmental Engineering, University of Alberta, Edmonton, Alberta T6G2W2, Canada. Tel: 780-492-8293; E-mail: aulrich@ualberta.ca

Word Count: 248 with 3 figures and 2 tables.

Page S4. Table S1: Concentration for non-metal and trace metal in the surrogate pond water Page S5. Table S2: List of primers and conditions used for DsrAB and MCR PCR amplification Page S6. Table S3: Relative abundance of selected sulfur reducing bacteria and methanogens Page S7. Figure S1: Dissolved organic carbon, and carbon dioxide measurements for the PW and SPW groups Page S8. Figure S2: Carbonyl sulfide, butyl mercaptan, and thiofuran concentrations for the PW and SPW groups

Page S9. Figure S3: 2-methylthiophene, 2-methylthiophene, 2,5-dimethylthiophene concentrations for the PW and SPW groups

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Detailed condition for Gas analysis

CH4 and CO2 productions in the mesocosms were measured by removing 50 μL headspace gas

and analyzing it with a 7890A gas chromatograph with a thermal conductivity detector (GC-

TCD) equipped with Supelco, 80/100 Hayesep Q column, 10 FT x 1/8 IN SS. Helium 5.0

(Praxair) was the carrier gas. Method parameters were the following: Inlet temperature and total

flow (200 oC and 15 mL/min); detector temperature and reference flow (200 oC and 30 mL/min);

and oven temperature was 25 oC (hold 3 minutes).

For sulfur gas analysis of hydrogen sulfide, carbonyl sulfide, thiofuran, butyl mercapatan, 2-

methylthiophene, 3-methylthiophene, and 2,5-dimethylthiophene, RSCs were analyzed using a

7890A GC and an Agilent Technologies 355 sulfur chemiluminescence detector (GC-SCD)

equipped with a DB-1 column from Agilent Technologies (part no. 123-1035). Hydrogen 5.0 and

Ultra zero air from Praxair were used for the SCD. Method parameters were the following: Inlet

temperature, total flow and split ratio (210 oC, 15 mL/min, 5:1); H2 flow rate, 39 sccm; Air flow

rate, 59.7 sccm; oven temperature, 30 oC for 3 mins; and ramp 15 oC/min to 185 oC. RSCs were

measured by injecting 5 mL gas samples into the SCD. If dilution was required, headspace gas

was diluted in 40 mL EPA vials filled with 5.0 nitrogen gas and sealed with Mininert caps. The

sulfur gas standard (Air Liquide) has the following component concentrations: hydrogen sulfide

(151 ppmv); carbonyl sulfide (5.09 ppmv), thiofuran (15.3 ppmv), butyl mercaptan (10.1 ppmv),

2-methylthiophene (91.6 ppmv), 3-methylthiophene (40.7 ppmv), and 2,5-dimethylthiophene

(61.1 ppmv).

Sulfate Reduction Rates Calculation

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The averaged SO42- concentration measurement was converted from mg/L to mol/L. Moles of

SO42- was calculated by multiplying SO4

2- concentration by the water volume inside the

mesocoms. The SO42- change (in moles) was then divided by the volume of MFT in mesocoms

and days between samples taken.

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Illumina MiSeq sequencing

Total metagenomic DNA was submitted to PCR amplification of the V4 hypervariable regions of

bacterial 16S rRNA genes, using the primers 515F (5ʹ-GTGCCAGCMGCCGCGGTAA-3ʹ) and

806R (5ʹ-GGACTACVSGGGTATCTAAT-3ʹ). Both forward and reverse primers were tagged

with adapter, pad and linker sequences. Each barcode sequence (12 mer) was added to the

reverse primer for pooling multiple samples in one run of MiSeq sequencing. The PCR

conditions were 95 °C for 3 min, 30 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min

of extension, followed by 72 °C for 5 min. PCR products were purified and combined in

equimolar ratios with the quantitative DNA binding method in order to create a DNA pool that

was further used for sequencing from the adaptor. The 16S rRNA gene fragments were

sequenced using the Illumina Miseq platform following the manufacturer procedure. The

sequences were deposited in SRA within the study with accession number PRJ. Sequences were

sorted out according to their barcode tags and trimmed to remove sequencing adaptors and

primers. After removing the low quality sequences, operational taxonomic units (OTUs) were

defined and assigned to taxa using UPARSE pipeline. The community matrix was normalized

with the DESeq package. The bacterial communities were ordinated with non-matric

multidimentsion scaling (NMDS) based on Bray-Curtis distance matrix using phyloseq.

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Table S1: Concentration for non-metal and trace metal in the surrogate pond water

Non-metal Compound

Concentration

(mg/L) Trace MetalConcentration (mg/L)

NH4Cl 16.4 Cr 0.0064KCl 28.1 Mn 0.0407NaCl 584.4 Co 0.0013MgSO4 56.7 Ni 0.0095Na2SO4 552.0 Cu 0.0054NaHCO3 1308.2 Zn 0.0071CaCl2 57.5 As 0.0132NaF 6.5 Se 0.0053KBr 0.5 Cd 0.0001SrCl26H2O 2.4 Sb 0.0015LiCl 1.7 Ba 0.3653

Mo 0.1180Ag 0.0001Ba 2.8900Si 3.0000

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Table S2: List of primers and conditions used for DsrAB and MCR PCR amplification.

  Primers Condition

DsrDSR1F - ACS CAC TGG AAG CAC G

DSR4R - GTG TAG CAG TTA CCG CA

28 cycle (98 °C-10s, 55 °C-10s, 72 °C-

1min)

mcr

A

F – TAY GAY CAR ATH TGG YT

R – ACR TTC ATN GCR TAR TT

38 cycle (98 °C-10s, 50 °C-30s, 72 °C-

30s)

16S

F –

TCGTCGGCAGCGTCAGATGTGTATA

A

GAGACAGCCTACGGGNGGCWGCA

G

R –

GTCTCGTGGGCTCGGAGATGTGTAT

AA

GAGACAGGACTACHVGGGTATCTA

ATCC

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Table S3: Relative abundance of selected sulfur reducing bacteria and methanogens.

   Desulfobulbaceae Desulfobacteraceae Desulfuromonadaceae

Desulfomicrobiaceae Methanoregulaceae Methanotrichaceae

 MFT week 0 1.74E-02 9.93E-03 1.35E-02 1.25E-03 2.70E-02 1.58E-02

Week 5

MFT-PW 7.37E-02 2.45E-02 1.40E-02 9.20E-03 4.92E-02 1.74E-02MFT-PW-0.8% 1.01E-01 2.61E-02 1.41E-02 6.32E-03 6.69E-02 2.03E-02MFT-SPW 6.33E-02 2.64E-02 2.20E-02 1.33E-02 3.74E-02 3.90E-02MFT-SPW-0.2% 6.87E-02 4.96E-02 6.79E-03 2.21E-03 1.08E-01 5.07E-02MFT-SPW-0.8% 6.25E-02 2.86E-02 2.03E-02 3.44E-02 5.12E-02 4.82E-02MFT-SPW-1.5% 8.19E-02 2.25E-02 1.68E-02 1.09E-02 4.38E-02 3.35E-02

Week 11

MFT-PW 1.11E-01 2.73E-02 1.13E-02 9.19E-03 7.15E-02 4.85E-02MFT-PW-0.8% 7.15E-02 2.66E-02 5.85E-03 4.39E-03 1.03E-01 5.65E-02MFT-SPW 4.25E-02 2.97E-02 1.11E-02 2.77E-03 7.27E-02 3.73E-02MFT-SPW-0.2% 7.87E-02 1.91E-02 1.43E-02 4.59E-03 6.00E-02 1.61E-02MFT-SPW-0.8% 2.69E-02 1.37E-02 2.41E-02 3.27E-02 3.57E-02 1.39E-02MFT-SPW-1.5% 7.02E-02 2.08E-02 1.77E-02 1.80E-02 4.11E-02 1.65E-02

Values indicate abundance in percentage of total reads.

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Figure S1: Dissolved organic carbon, and carbon dioxide measurements for the PW and SPW groups over the 11-week study period. The values for the methane measurement, MFT (PW, SPW, and SPW-0.2%) are averages from duplicate measurements. For all other mesocosms, only one measurement is represented. Filled circles: PW or SPW +0.8% diluent; open diamonds: +MFT; open triangle: +MFT/+0.2% diluent, open square: +MFT/+0.8% diluent; x: +MFT/+1.5% diluent

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Figure S2: Carbonyl sulfide, butyl mercaptan, and thiofuran concentrations for the PW and SPW groups over a 14-week study period. The values for butyl mercaptan (PW-0.8% at W0) and thiofuran (PW-0.8%, all; MFT-SPW-0.8% at W6; MFT-PW-0.8% at W4 and W14; MFT-SPW-0.8% at W4; and MFT-SPW-1.5% at W4) represent one measurement. All other values are averages from duplicate measurements and the error bars (where visible) represent plus and minus one standard deviation. Filled circles: PW or SPW +0.8% diluent; open diamonds: +MFT; open triangle: +MFT/+0.2% diluent, open square: +MFT/+0.8% diluent; x: +MFT/+1.5% diluent

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Figure S3: 3-methylthiophene, 2-methylthiophene, 2,5-dimethylthiophene concentrations for the PW and SPW groups over a 14-week study period. Filled circles: PW or SPW +0.8% diluent; open diamonds: +MFT; open triangle: +MFT/+0.2% diluent, open square: +MFT/+0.8% diluent; x: +MFT/+1.5% diluent

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