1. Xiaofang Wei 1 Xingli Li 2 Keyu Liu 3 Yuehui She 4 Jing Wang 1 1 China University of Petroleum 2 GRI, Jiangsu Oilfield 3 CSIRO Petroleum 4 Yangtz University MEOR Using Facultative Anaerobic Indigenous Consortia and Bio-surfactants

2. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Acknowledgements CSIRO WfO Flagship Biochemical Engineering Laboratory, CUP, Beijing Yijng Luo, Mang Lu, Peiwen Zheng, Zhongzhi Zhang The Langfang Research Institute of Petroleum Exploration and Development, PetroChina Core-flooding technical support

3. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Presentation Outline Background of the MEOR study Nutrient stimulation MEOR screening and assessment Bacteria selection and metabolite analysis Oil spreading and blood agar lysis methods Emulsification Interfacial tension (IFT) Core flooding experiments Experimental procedure Results Conclusions and Future work

4. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Enhanced Oil Recovery Unrecovered oil range from 1-2 trillion barrels

5. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia World Average Oil Price

6. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Oil Consumption (BP Statistical Review, 2006) Australia:0.9 mbbl/day China:7.0 US:20.6 World:82.5 US 1 cubic km oil / year Australia China United States 1 km l l

7. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia EOR Portfolio NON-THERMAL Miscible Chemical Imm. Gas Drives Other Flue Gas CO 2 Inert Gas FOAM MEOR Surfactant Polymer Alkaline Enriched Gas Drive Slug Process N 2 Miscible CO 2 Miscible Alcohol ASP Micellar Emulsion Vaporizing Gas Drive MEOR is a rapidly growing EOR technique currently under laboratory research or field investigation

8. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia MEOR Mechanisms and Strategies Acids, gas, solvents production Commonly made by carbohydrate fermentation Inject carbohydrates (molasses, etc.) Selective plugging Create biofilms, cell mass and/or mineral precipitates Any nutrient could work: use nitrate as electron acceptor If reservoir has high divalent cation concentration, CO 2 production may stimulate carbonate formation Hydrocarbon degradation Inject electron acceptor and/or limiting nutrients (N, S, P, metals) Biosurfactant production Emulsifiers Periodic cycles of nutrient-excess and nutrient limitation

9. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Microbially Converting CO2 to CH4 in Depleted Reservoirs (from Maeda, 2009)

10. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Implementing MEOR (from McInerney,2009) Not all reservoirs are candidates for MEOR Environmental conditions limit microbial growth Recommendations: Temperature: <80 o C pH: 5-9 Salinity: <10% Depth: <2500 m API gravity: > 15 o Residual oil saturation: >25%

11. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Background of the Reservoir Investigated Characteristics of the Huatugou Oil Reservoir, Qinghai Oilfield, China Production history: 1958-present EOR: Water-flooding Formation Water Salinity <2x10 4 ppm Reservoir P/T: 45 ℃ /22 MPa Fm water pH≤7 Low sulfate concentration

12. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Microbial Screening Hydrocarbon Degrading Bacteria (HBD) Microbes population>10 5 cells/ml (Bottle test) Sulphate Reducing Bacteria (SRB) Not Detected (PCR)

13. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia MEOR Screening The Huatugou reservoir is a good candidate for MEOR Relatively high microbe population: >10 5 (Cell counting) Low formation Water Salinity: <2x10 4 ppm Low reservoir T: 45 ℃ Suitable formation water chemistry: pH≤7 Low corrosion risk: no SRB detected

14. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Nutrient Stimulation Proper nutrient (Carbon Source: Reservoir oil) Target facultative anaerobic indigenous consortia Cell Population is up to 10 8 cells/ml Minor IFT and Viscosity changes Gas production observed

15. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Bio-surfactant: species selection and identification Two strains were selected and named as BIOS682- 1 and BIOS682-2 Initial screening by the blood agar lysis method: positive Screening using the oil spreading method Pure culture clear zone diameters : 4.5 cm and 4.8 cm Mixed culture clear zone diameter: 5.0 cm Species identification using 16s RNA BIOS682-1: Brevibacillus agri (99% similarity) BIOS682-2: Brevibacillus levicki (95%, a possible new species)

16. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Bio-surfactants: species culture and assessment Mixed culture of two selected strains 5ml oil/50ml-medium cultured for 3 weeks at 45 ℃ Under facultative anaerobic condition (headspace filled with N 2 ) Oil-water separation Oil was centrifuged at 5000 rpm for 10 min at room temperature Oil was extracted by using CCl 4 (oil in water) The surface tension Equipment: Contact Angle analyzer (DCA322) Emulsification (Culture and reservoir oil 1:1 v/v) Emulsification maintained for 100 hrs after 15 min u/s bathing The ratio between emulsified oil and water measured

17. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Bio-surfactant producing species: culture and assessment Surface tension reduction: 24% Emulsification improvement: 14%

18. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Bio-surfactant analysis Bio-surfactant of lipopeptide extraction protocol centrifuged twice at 4 ° C @10,000 rpm for 20 min the supernatant was mixed with 6mol HCL @ pH=2 (24 hrs 4 ° C) The floccules were collected using centrifuge (4 ° C @10,000 rpm for 20 min) The settlings were washed using acidic solution (pH=2), then dissolved in weak alkali solution (pH=8) Extracted with DCM and methanol solution (v/v 3:1) Dried under vacuum at low temperature The primary products were dissolved in alkali solution (pH=8) The extraction was then dried under vacuum

19. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Bio-surfactant analysis Thin Layer Chromatography (TLC) Initial screening Liquid Chromatography-Mass Spectrometry (LC-MS). The purified product was hydrolyzed for 14 hrs at 110 ℃ with 6N HCl in N2 chamber Equipment: Agilent 1100 system  -Amino acid

20. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Bio-surfactant analysis Thin Layer Chromatography (TLC) lipopeptide bio-surfactant (Pink): Q ualitative analysis Solution used is a mixture of n-butanol/alcohol/water(4:1:1) Metabolit e Standard Sample

21. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Bio-surfactant analysis lipopeptide bio-surfactant-Amino Acid (Yellow) Metabolit e Standard Sample

22. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Bio-surfactant analysis Amino Acid contents in the metabolites

23. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia 4. Core-flooding Experiment Core-flooding experiment design: T/P: 45 ℃ /22 MPa Water-cut: 70% Incubation time: 3 days Injected volume: 2.5 PV of Cultured solution Oil Water Microbes Collector Core Holder

24. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Synthetic Core Plugs for Core Flooding Experiments Material: oil zone sandstone recovered from the producing well, Disintegrated  Cleaned Sieved to 30, 80 and 120 meshes, respectively A two-layer heterogeneous sand packs with different permeabilities was constructed Layer 1: 2:2:1 mix of 30-80, 80-120 and >120 meshes Layer 2: 1:1 mix of 30-80 and 80-120 meshes

25. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Core-flooding Experiment η ＝ (B － A) － C B: final oil recovery % A: oil recovery % from 0.5 pv nutrient solution injection C: oil recovery % (Blank) from 0.5 pv synthetic formation water injection η: incremental oil recovery (%) Core plug properties

26. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Results of core-flooding

27. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Conclusions The Huatugou reservoir in the Qinghai Oilfield is a good candidate for MEOR The indigenous facultative anaerobic flora from the reservoir was stimulated by adding proper nutrients to promote their activities The two indigenous aerobic species selected can co- metabolize well and produced more bio-surfactant than from a single strain in the reservoir The two species identified by 16s RNA are Brevibacillus agri (BIOS682-1); and a possible new species in genus of Bacillus brevis (BIOS682-2).

28. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Conclusions The metabolites of BIOS682 contain low concentration of amino acids An 11% incremental recovery was achieved using a combined microbial and biosurfactant solution injection. The facultative anaerobic flora play an important role in peeling the oil form rock or breaking down the large oil drops. The use of cultured solution with bio-surfactant and other metabolites is much more effective than the use of the indigenous microbes alone in recovering residual oil.

29. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Future work In-situ experiments (Field Trials) Further analysis of the metabolites

30. 30 th IEA Workshop and Symposium on EOR Sept. 21-23, 2009, Canberra, Australia Thank You Dr Keyu Liu Research Team Leader CSIRO Petroleum Keyu.Liu@csiro.au Dr Xiaofang Wei Research Institute of Petroleum Exploration and Development, PetroChina weixfanf@163.com