Exploration and Production Technologies

Bio-Engineering High Performance Microbial Strains for MEOR by Directed-Protein-Evolution Technology


Project Goal
The goals of this project are to 1) apply advanced bio-engineering methods (such as genetic manipulation) to induce bacteria that naturally make biosurfactants do so at a much higher, commercially useful rate; and 2) implant the genetic information for rapid biosurfactant production into microbes adaptable in an oil reservoir environment.

California Institute of Technology, Pasadena, CA

This project is currently in the third and final phase of proposed 3-year research period. Significant progress has been made since the project last update, including the following accomplishments:

  • Researchers have successfully engineered the new mutant strains P. aeruginosa PEER02 and E. coli TnERAB so they can produce rhamnolipid biosurfactants.
  • Core flooding tests showed that rhamnolipids produced by the researchers’ engineered bacteria are effective agents for microbial enhanced oil recovery (MEOR). At 250 ppm rhamnolipid concentration from P. aeruginosa PEER02, 42 percent of the remaining oil after waterflood was recovered. These results were therefore important for considering the exploration of the studied rhamnolipids as EOR agents.
  • The engineered P. aeruginosa PEER02 strain can produce rhamnolipids with different carbon sources as substrate. Interfacial tension analysis (IFT) showed that different rhamnolipids from different substrates gave different performance.
  • Through the methodology of synthetic biology and metabolic engineering, researchers engineered E. coli strains harboring various gene combinations from P. aeruginosa and successfully produced either mono-rhamnolipids or di-rhamnolipids (one or two head groups).

This project benefits the industry by identifying a wider spectrum of types of surfactant products that may be useful for EOR. In particular, bio-based surfactant alternatives offer new (and perhaps better) choices for an EOR project. These chemicals are more environmentally friendly and can come from renewable resources.

The State and the public benefit of this research is that it promotes MEOR, and thereby a method to increase domestic oil supply. There is also a general benefit because the project approach is a successful example for other researchers to follow. Other oilfield or industrial chemicals may be created using bioprocesses that will produce a product that costs less, and is environmentally friendly.

This 3-year project began in October 2004 as an effort to improve the cost-effectiveness of MEOR. The motivation for this study was that the oil industry had a history from the previous “boom” period of the late 1970s and 1980s of developing MEOR in the laboratory and having some moderately successful technical field tests. However, the economics of MEOR prevented widespread commercial deployment of the technology, due in part to the high cost of the nutrients to maintain the microbes in-situ and the low production rate of biosurfactant.

The current project seeks surface processes to manufacture biosurfactant rates efficiently from waste feed streams so that these chemicals can be cost-competitive with synthetic surfactants. Implanting this surfactant-making ability in microbes adapted to oil makes feasible an in-situ MEOR process that requires little operator maintenance.

Project researchers have:

  • Cloned genes involved in the rhamnolipid and surfactin bio-synthesis.
  • Successfully produced rhamnolipids in both P. aeroginosa PAO1-RhlA- strain and P. fluorescens ATCC15453 strain, with an increase of 55-fold to 175-fold in production compared with wild-type bacteria strain.
  • Successfully engineered E. coli strains that can produce the rhamnolipids.
  • Established several methods, such as colorimetric agar plate assay, colorimetric spectrophotometer assay, and oil-spreading assay, to detect and screen rhamnolipid and surfactin production.
  • Characterized the behavior of the rhamnolipid and surfactin as EOR agents by surfactant adsorption assay, interfacial tension assay, and wettability tests using calcite flotation methods.
  • Characterized the behavior of the rhamnolipid as EOR agents by core flooding experiments. Engineering the rhamnolipid at an injected concentration as low as 250 ppm recovered 42 percent of remaining oil after waterflooding.

Current Status (December 2008)
This project is completed and the final report is available below under "Additional Information".

This project was selected in response to the DOE Oil Exploration and Production solicitation DE-PS26-04NT15450-3B, with a focus on Enhanced Oil Recovery.

Project Start: October 1, 2004
Project End: September 30, 2007

Anticipated DOE Contribution: $766,786
Performer Contribution: $191,696 (25 percent of total)

Contact Information
NETL – Traci Rodosta (Traci.Rodosta@netl.doe.gov or 304-285-1345)
Cal Tech - William Goddard (wag@wag.caltech.edu or 626-395-2731)
Cal Tech - Xiangdong Fang (xdfang@peer.caltech.edu or 626-858-5077)

Additional Information 
Final Project Report [PDF-1.32MB]

Fang X., Wang Q., Bai B., Liu X., Shuler P., Tang Y. and William G.A., “Engineering Rhamnolipid Biosurfactants as Agents for Microbial Enhanced Oil Recovery,” accepted for presentation at the 2007 SPE International Symposium on Oilfield Chemistry held in Houston, TX, February 28–March 2, 2007.

Wang Q., Fang X, Bai B., Liang X., Shuler P., Goddard W.A. and Tang Y., “Engineering bacteria for production of rhamnolipid as an agent for enhanced oil recovery,” submitted to Biotechnology and Bioengineering, January 2007.

Second annual project report to DOE, December 2006. First annual project report to DOE, October 2005.

IFT analysis of rhamnolipid in various conditions. (a) Profile of IFT of different concentration of rhamnolipid in water. (b) Effects of pH on IFT of rhamnolipid. Diamond: no NaCl; Square: 2% NaCl; Triangle: 8% NaCl. (c) Effects of Salinity on IFT of rhamnolipid. Diamond: pH 6; Square: pH 5; Triangle: pH 4. (d) Effects of temperature on IFT of rhamnolipid. Diamond: Rhmanolipid in pH 4, 1%NaCl; Square: pH 5, 2%NaCl; Triangle: pH 6, 8%NaCl.

Oil recovery test of a waterflooded sand pack core by rhamnolipid flooding. (a) Profile of oil recovery (Triangle) and IFT (Circle) during flooding. b) Water cut (Square) and cumulative oil recovery (Diamond). (I) rhamnolipid flooding; (II) Brine flooding. One pore volume was 85 ml brine solution (50mM citrate-Na2HPO4, 2% NaCl, pH 5.0 buffer). The porosity and brine permeability of the sand-packed core in this experiment was 45% and 17.9 Darcies, respectively.


IFT analysis, main congeners and their relative abundance of rhamnolipids from various sources.

Effects of pH and salinity on the interfacial tension (IFT) value of rhamnolipids produced by recombinant strain PAAB06. Each strain produces a different rhamnolipid structure that has a different optimal salinity and pH to create its lowest IFT. Data shown for n-octane as the oil phase and at 30 C.

Oil recovery of initial oil in-place during surfactant flood and water flood. (500 mg/l biosurfactant). The sand pack absolute permeability is about 20 D with a pore volume of 85 ml. One PV of brine was injected into the core, and then 3-PV of 500 mg/l rhamnolipid was injected into the core to flush remaining oil; finally, more than 10-PV of brine was injected. The initial water injection recovered 62.9% OOIP, 3-PV surfactant injection increased the oil recovery to 67.6% OOIP. Water flood after surfactant flood further increased oil recovery to 80.8% OOIP. Surfactant flood and then water flood recovered about 48.3% of remaining oil.