in Microbial Enhanced Oil Recovery

Super Oil Magician Team Nankai in Microbial Enhanced Oil Recovery

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

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

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

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

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

Solution--Surfactants???

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

Surface Activities Promote capillarity by reducing the water surface tension.

Surface Activities Thus leading the water into the small cavities.

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

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

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

Strategy An engineered anaerobic rhamnolipid-producing bacterial strain

Mechanism Attracted Growing& Producing Oil Emulsified

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

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

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

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

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

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.

Evaluation of Our Super Oil Magician

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

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

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

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

Plasmid construction recombinant expression plasmid

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

Transformation of E.coli Agarose electrophoresis result 900 bps

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

Fermentation products reduced surface tension of the medium. Liquid Bacteria growth Surface tension Emulsifying ability Pure culture medium - 68.20 - E.coli wildtype +++ 63.75 - E.coli with pbbr1 empty vector +++ 65.24 - E.coli with pbbr1- rhlabri +++ 39.34 + The number of + reflects the degree of each parameters.

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

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

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

Transform with pbbr1-mapple for test A fluorescence protein

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

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

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

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

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

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.

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

Physical Simulation of Oil Displacement Rhamnolipid solution

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

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

Artificial Core permeability The artificial core container porosity The artificial core

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

The oil recovery rate of rhamnolipid

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

Super Oil Magician

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

Lack of materials?

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

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

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

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

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

Project Introduction NKU Oil microbe TJU Transfibre Team NKU Team TJU

Academic Salon Details, theories. Application, igem Jamboree

NKU igem TEAM & TJU igem TEAM MUTUAL ASSISTANCE

Mutual Assistance SDS-PAGE Art Design

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.