ARTICLE Engineering Bacteria for Production of Rhamnolipid as an Agent for Enhanced Oil Recovery Qinhong Wang, Xiangdong Fang, Baojun Bai, Xiaolin Liang, Patrick J. Shuler, William A. Goddard III, Yongchun Tang Power, Energy and Environment Research (PEER) Center, Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125; telephone: 626-858-5077; fax: 626-858-9250; e-mail: xdfang@peer.caltech.edu Received 23 January 2007; revision received 5 April 2007; accepted 6 April 2007 Published online 7 May 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21462 ABSTRACT: Rhamnolipid as a potent natural biosurfactant has a wide range of potential applications, including enhanced oil recovery (EOR), biodegradation, and bio- remediation. Rhamnolipid is composed of rhamnose sugar molecule and b-hydroxyalkanoic acid. The rhamnosyltrans- ferase 1 complex (RhlAB) is the key enzyme responsible for transferring the rhamnose moiety to the b-hydroxyalkanoic acid moiety to biosynthesize rhamnolipid. Through transposome-mediated chromosome integration, the RhlAB gene was inserted into the chromosome of the Pseudomonas aeruginosa PAO1-rhlA  and Escherichia coli BL21 (DE3), neither of which could produce rhamnolipid. After chromosome integration of the RhlAB gene, the constitute strains P. aeruginosa PEER02 and E. coli TnERAB did produce rhamnolipid. The HPLC/MS spectrum showed that the structure of purified rhamnolipid from P. aeruginosa PEER02 was similar to that from other P. aeruginosa strains, but with different percentage for each of the several con- geners. The main congener (near 60%) of purified rhamnolipid from E. coli TnERAB was 3-(3-hydroxydeca- noyloxy) decanoate (C 10 –C 10 ) with mono-rhamnose. The surfactant performance of rhamnolipid was evaluated by measurement of interfacial tension (IFT) and oil recovery via sand-pack flooding tests. As expected, pH and salt concentration of the rhamnolipid solution significantly affected the IFT properties. With just 250 mg/L rhamnolipid (from P. aeruginosa PEER02 with soybean oil as substrate) in citrate–Na 2 HPO 4 , pH 5, 2% NaCl, 42% of oil otherwise trapped was recovered from a sand pack. This result suggests rhamnolipid might be considered for EOR applications. Biotechnol. Bioeng. 2007;98: 842–853. ß 2007 Wiley Periodicals, Inc. KEYWORDS: rhamnolipid; biosurfactant; transposome; chromosomal insertion; interfacial tension; enhanced oil recovery Introduction Surfactants pervade our everyday lives. Some surfactants, referred to as biosurfactants, are produced by bacteria or yeast from various substrates including sugars, glycerol, oils, hydrocarbons and agricultural wastes (Lin, 1996). Biosurfactants are classified as glycolipids, lipopeptides, phospholipids, fatty acids, neutral lipids, and polymeric or particulate compounds (Desai and Banat, 1997). The hydrophobic portion of the molecule is long-chain fatty acids, hydroxyl fatty acids or a-alkyl-b-hydroxyl fatty acids. The hydrophilic moiety can be a carbohydrate, amino acid, cyclic peptide, phosphate, carboxylic acid or alcohol. Biosurfactants have been receiving increasing attention as a result of their unique properties, i.e., mild production conditions, lower toxicity, and higher biode- gradability, compared to their synthetic chemical counter- parts (Rosenberg and Ron, 1999). Thus, it may be possible to reduce the environmental impact by replacing chemical additives with biosurfactants. The initial focus and most current industrial interest in biosurfactants are toward applications in the oil industry to aid in the clean up of oil spills and for enhanced oil recovery from mature oil Correspondence to: X. Fang Contract grant sponsor: Department of Energy Contract grant number: DE-FC26-04NT15525 This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/0006-3592/suppmat. 842 Biotechnology and Bioengineering, Vol. 98, No. 4, November 1, 2007 ß 2007 Wiley Periodicals, Inc.