in Microbial Enhanced Oil Recovery

Transcription

1 Super Oil Magician Team Nankai in Microbial Enhanced Oil Recovery

2 Contents Introduction Design Construction of Plasmid Transformation of E.coli Transformation of Pseudomonas stutzeri Physical Simulation of Oil Displacement ishare Human Practice

3 Contents Introduction Design Construction of Plasmid Transformation of E.coli Transformation of Pseudomonas stutzeri Physical Simulation of Oil Displacement ishare Human Practice

4 Current Situation We use water drive to get most of the oil underground.

5 Problem I At the end of water drive, residual oil sticks in the small cavities and pores along the water pathway.

6 Problem I At the end of water drive, residual oil sticks in the small cavities and pores along the water pathway.

7 Solution--Surfactants???

8 Solution--Surfactants Surface Activities Interfacial Activities Emulsify Properties Environment Friendly Biosurfactant Rhamnolipid in Microbial Enhanced Oil Recovery

9 Surface Activities Promote capillarity by reducing the water surface tension.

10 Surface Activities Thus leading the water into the small cavities.

11 Interfacial Activities Break the water-oil interface and mix them by reducing interfacial tension. Shake

12 Emulsify Properties Stabilize the water-oil mixture, emulsification. ---Diesel ---Emulsified Diesel-Water Mixture ---Water Control Add Rhamnolipid

13 Problem II Shortages of Current Injection Methods Direct Injection(Ex situ) Costly Harmful to Environment Low efficiency In situ Production Aerobic Microorganism Anaerobic Surroundings Oxygen High Low

14 Strategy An engineered anaerobic rhamnolipid-producing bacterial strain

15 Mechanism Attracted Growing& Producing Oil Emulsified

16 Effects Locally release rhamnolipid Low water surface tension Lead the water in and wash the oil off

17 Contents Introduction Design Construction of Plasmid Transformation of E.coli Transformation of Pseudomonas stutzeri Physical Simulation of Oil Displacement ishare Human Practice

18 Suitable Microorganism for In-situ Rhamnolipid Production - Pseudomonas stutzeri Naturally lives in oil reservoirs! No need of oxygen! Feed on oil! Pseudomonas stutzeri

19 Modifying the Metabolic Pathways - Adding the Ligation Device rhlabri + Ligation Device rhlabri Rhamnosyltransferase (product of rhabri) Fatty acid synthetic pathway Rhamnose Fatty acid Rhamnose producing pathway Rhamnolipids

20 The Ligation Device rhlabri rhla Pseudomonas Source genes related to rhamnolipid synthesis rhlb rhlr rhli rhlc OXYGEN Pseudomonas aeruginosa

21 The Ligation Device rhlabri More bacteria reproduced Higher concentration of C4-HSL More copies of A&B genes activated Greater rhamnolipid production Especially in oil-rich cavities! Quorum Sensing Originally induced by R&I in P. aeruginosa C4-HSL, an autoinducer which can be secrete by one bacterium into the environment and sensed by others in vicinity.

22 Evaluation of Our Super Oil Magician

23 Evaluation of Our Super Oil Magician Measurements: Emulsifying Ability Emulsifying Ability weak (left), medium (middle), strong (right)

24 Evaluation of Our Super Oil Magician Measurements: Emulsifying Ability Surface Tension testing of surface tension

25 Evaluation of Our Super Oil Magician Measurements: Emulsifying Ability Surface Tension Simulate Oil Recovery Rate The Artificial Core: tunnel filled with grits and crude oil

26 Contents Introduction Design Plasmid Construction Transformation of E.coli Transformation of Pseudomonas stutzeri Physical Simulation of Oil Displacement ishare Human Practice

27 Plasmid construction recombinant expression plasmid

28 Contents Introduction Design Construction of Plasmid Transformation of E.coli Transformation of Pseudomonas stutzeri Physical Simulation of Oil Displacement ishare Human Practice

29 Transformation of E.coli Agarose electrophoresis result 900 bps

30 Engineered E.coli strain produced rhamnolipid. High Performance Liqid Chromatography (HPLC) Thin-LayerChromatography (TLC)

31 Fermentation products reduced surface tension of the medium. Liquid Bacteria growth Surface tension Emulsifying ability Pure culture medium E.coli wildtype E.coli with pbbr1 empty vector E.coli with pbbr1- rhlabri The number of + reflects the degree of each parameters.

32 Fermentation products enhanced emulsifying ability of the medium. Control E.coli with pbbr1-rhlabri

33 Contents Introduction Design Construction of Plasmid Transformation of E.coli Transformation of Pseudomonas stutzeri Physical Simulation of Oil Displacement ishare Human Practice

34 Empty vector? With rhlabri pbbr1 empty vector pbbr1-rhlabri CaCl₂ heat shock Electroporation

35 Transform with pbbr1-mapple for test A fluorescence protein

36 mapple expresses in P.stutzeri 10-5 pbbr1-mapple expression system of pbbr1 can work well in P.stutzeri

37 rhlc had been ignored in our previous construction rhla rhlb Rhamnosyltransferase I rhl-related genes rhlr rhli Regulate the transcription rhlc PA1131

38 PA1131, a downstream gene of rhlc might be involved in exportation of rhamnolipid major facilitator superfamily transporter PA1131

39 The accumulation of rhamnolipids might affect the growth of P. stutzeri PA1131 major facilitator superfamily transporter Secondary metabolites might affect growth The absence of PA1131 might affect the secretion of rhamnolipid

40 Future work Clone the rhlc and PA1131 gene Construct a recombinant expression plasmid Transform the recipient bacterium

41 Due to limited time, the engineered strain is still under construction. However, we are still able to estimate the effects of producing rhamnolipid in situ on oil recovery through our physical model.

42 Contents Introduction Design Construction of Plasmid Transformation of E.coli Transformation of Pseudomonas stutzeri Physical Simulation of Oil Displacement ishare Human Practice

43 Physical Simulation of Oil Displacement Rhamnolipid solution

44 Complicated Factors Temperature Viscosity of oil Moisture content Paraffin content Oil reservoir

45 Simulate the oil reservoir in lab simulate simplify Oil reservoir Sing-Well Simulation Device

46 Artificial Core permeability The artificial core container porosity The artificial core

47 Simulation procedure Device prepared Empty pore Loaded with oil Pore filled with oil Injected water Pore filled with water Injected rhamnolipid solution An artificial core Pore filled with rhamnolipid solution

48 The oil recovery rate of rhamnolipid

49 Results in simulation experiment & Real effect In situ production of rhamnolipid

50 Super Oil Magician

51 Contents Introduction Design Construction of Plasmid Transformation of E.coli Transformation of Pseudomonas stutzeri Physical Simulation of Oil Displacement ishare Human Practice

52 Lack of materials?

53 Wiki platform promotes sharing Bio-bricks---iGEM Bacterial strains Materials Antibodies Cell lines Project Physical modeling Parts Practice Team More

54 Add a special part on wiki---resource sharing Resource Sharing Lable the materials and their sources

55 Contents Introduction Design Construction of Plasmid Transformation of E.coli Transformation of Pseudomonas stutzeri Physical Simulation of Oil Displacement ishare Human Practice

56 Human Practice Exchange Conference Project Introduction Academic Salon Mutual Assistance Test Protein

57 Active igem collaboration NKU igem TEAM & TJU igem TEAM EXCHANGE CONFERENCE

58 Project Introduction NKU Oil microbe TJU Transfibre Team NKU Team TJU

59 Academic Salon Details, theories. Application, igem Jamboree

60 NKU igem TEAM & TJU igem TEAM MUTUAL ASSISTANCE

61 Mutual Assistance SDS-PAGE Art Design

62 We designed project together. We constructed plasmids together. We did experiments together. Thanks We enjoyed the learning experience. We designed our Wiki together. And, today, we stand here together, to show what we have done.