Derisking Offshore Oil Exploration with Lipidomics

1. Derisking Offshore Oil Exploration with Lipidomics G. Todd Ventura

2. Derisking Offshore Oil Exploration with Lipidomics Bioassay 1 Aerobic HC Oxidizers Bioassay 2 Anaerobic HC Oxidizers Bioassay 3 Endospores Lipidomics Petroleum Geochemistry

3. How it works – Lipidomics and IPLs Lipidomics – The large-scale study of the structure, pathways and networks of cellular lipids in biological and geochemical systems.

4. How it works – Lipidomics and IPLs Extracellular fluid Glycolipids Glycoprotein Bilayer and hydrophobic core Phospholipid fatty acids Peripheral protein Cytoplasm

5. Intact Polar Lipids Bacterial IPLs – Archaeal IPLs – Head group Glycerol Aliphatic tail Head group ether Isoprenoid tail

6. Diversity of IPLs Aktas et al., 2017 Fredricks et al., 2004

7. Other Lipids of Consideration • Fossil Lipids: • Core lipids • Hydrocarbon biomarkers

8. Research Strategy • Proof of concept • Validation

9. Research Strategy – Proof of Concept Carl has collected frozen piston core samples being stored at Bedford Institute of Oceanography (BIO). Dr. Carl Peters Mitacs Postdoctoral Fellow

10. Research Strategy – Proof of Concept • Matrix matched reference material – Bay of Fundy estuary mud • Total Lipid Extract (TLE) recovery: • Number of Repeats = 6

11. Bay of Fundy Mud – Matrix Matched Reference Material (HILIC-uHPLC-1% TLE injected_qToF file 8159) Int. HPH-GDGT-0 1750 2G-OH-GDGT HPH-Cren 5 10 1G-OH-GDGT-0 ? ? ? ? ? 1500 1G-GDGT-0 DPG 1G-Cren 4 10 PG-DAG & PC-DAG Dimers 1250 Lyso-DPG 3 10 m/z 2G-DAG 1000 ? PI 2 10 PG-DAG BL 1G-DAG PC-DAG 750 1 10 PDME OL PE-DAG 500 0 10 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 Time [min] BL – characteristic fragment in MS2 of m/z 236.1492 1G-DAG – characteristic head group loss of 197 Da (sugar+NH4+) PDME - characteristic head group loss of 169.0504 Da PG - characteristic head group loss of 189.0402 Da PC – characteristic fragment in MS2 of m/z 184.0733 PE - characteristic head group loss of 141.0191 Da PME - characteristic head group loss of 155.0347 Da OL – characteristic fragment in MS2 of m/z 115.0866 PI - characteristic head group loss of 277 Da

12. Research Strategy – Proof of Concept Sample Sites Modified from Fowler and Webb (2017)

13. Research Strategy – Proof of Concept HC negative site HC positive site HC positive site

14. Research Strategy – Proof of Concept 2016-44 A‘A‘‘ (HILIC-uHPLC - 10% TLE injected_qToF file 8166) Int. 2G-2OH-GDGT 1750 2G-OH-GDGT 2G-GDGT-0 5 10 1G-2OH-GDGT-0 1G-OH-GDGT-0 1500 1G-GDGT-0 1G-Cren 4 10 1250 3 10 m/z 1000 2 10 750 1 10 500 0 10 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 Time [min]

15. Research Strategy – Proof of Concept 2016-42 4m (HILIC-uHPLC-10% TLE injected_qToF file 8165) Int. 2G-2OH-GDGT 1750 2G-OH-GDGT 2G-GDGT-0 5 10 1G-2OH-GDGT-0 1G-OH-GDGT-0 1500 1G-GDGT-0 1G-Cren 4 10 1250 3 10 m/z 1000 2 10 unknown bacterial (?) series 750 1 10 500 0 10 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 Time [min]

16. Research Strategy – Proof of Concept • Reproducible extraction method that is yielding lipids. • Extracts from HC positive Scotian shelf piston cores appear to be consistently greater than for a HC negative site. • The Scotian Shelf sediments have IPLs that are much less diverse than what we see for our reference standard. • Both HC positive and negative sites have Archaeal IPLs. • So far, the HC positive site also contains what are likely to be Bacterial IPLs.

17. Research Strategy – Proof of Concept • Reproducible extraction method that is yielding lipids. • Extracts from HC positive Scotian shelf piston cores appear to be consistently greater than for a HC negative site. • The Scotian Shelf sediments have IPLs that are much less diverse than what we see for our reference standard. • Both HC positive and negative sites have Archaeal IPLs. • So far, the HC positive site also contains what are likely to be Bacterial IPLs. Research Strategy – Validation

18. Research Strategy - Validation Dr. Casey Hubert and his group have conducted three sampling seasons. 2015 – Where, CCGS Hudson 2016 – Where, CCGS Hudson 2017 – Sydney Basin, RV Coriolis

19. Research Strategy - Validation 73 samples available • Representing: • 8 HC positive sites: Thermogenic • 2016-0042 • 2016-0032 • 2015-0006 Biogenic • 2016-0048 • 2016-0049 • 2015-0009 • 3 HC negative sites: Core Sampling Strategy

20. Research Strategy - Validation • Develop Scotian Shelf lipid library • Determine microbial substrate utilization via CSIA • Compare results to Calgary’s genomic data Int. 2G-2OH-GDGT 1750 2G-OH-GDGT 5 10 1G-2OH-GDGT-0 1G-OH-GDGT-0 1500 1G-GDGT-0 1G-Cren SRB 4 10 1250 3 10 m/z 1000 ANME-2 2 10 750 1 10 ANME-1 500 0 10 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 Time [min]

21. Conclusions • We believe we have the right strategy and support network to evaluate the use of IPLs as IHIs for offshore exploration in Nova Scotia. • Stay tuned for more!

22. Acknowledgements Collaborators: Casey Hubert, Jayne Rattray, Julius Lipp, Florence Schubotz, and Kai Uwe-Hinrichs. Funding Sources: - Canadian Research Chair - Canadian Foundation for Innovation – JELF - Offshore Exploration Research Association (Halifax, Nova Scotia, Canada) - Department of Energy Nova Scotia - Mitacs - SMU FGSR

23. Thank you

24. How it works – Lipidomics and IPLs

25. Derisking Offshore Oil Exploration with Lipidomics • We are working to generate lipid profiles extracted from piston core sediments collected at known seep sites to target subsurface living microbial communities that are actively degrading petroleum-based hydrocarbons. • This project aims to compare the sensitivity of lipidomics with that of the University of Calgary’s bioassay technology. • The lipid assignments for hydrocarbon oxidation are equally to be compared with the petroleum geochemistry data produced by APT Ltd. Canada.

26. Microbial Hydrocarbon Oxidation Contingent is the hypothesis working is that microbes are proliferating in seep sediments by anaerobically oxidizing petroleum forming hydrocarbons How does this look:

27. Microbial Hydrocarbon Oxidation