Colloids and Surfaces B: Biointerfaces 81 (2010) 397–405 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Structural characterization of a rhamnolipid-type biosurfactant produced by Pseudomonas aeruginosa MR01: Enhancement of di-rhamnolipid proportion using gamma irradiation Tayebe B. Lotfabad a , Habib Abassi b , Reza Ahmadkhaniha c , Reza Roostaazad a , Fatemeh Masoomi b , Hossein S. Zahiri b , Gholamreza Ahmadian b , Hojatollah Vali d , Kambiz A. Noghabi b,∗ a Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran b Department of Molecular Genetics, National Institutes of Genetic Engineering and Biotechnology (NIGEB), P.O.BOX 14155-6343, Tehran, Iran c Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14174, Iran d McGill Facility for Electron Microscopy Research, Montreal, QC, Canada article info Article history: Received 16 April 2010 Received in revised form 18 June 2010 Accepted 24 June 2010 Available online 23 July 2010 Keywords: Rhamnolipid Gamma irradiation Monorhamnolipid Di-rhamnolipid Pseudomonas aeruginosa MR01 abstract We previously reported that MR01, an indigenous strain of Pseudomonas aeruginosa, was able to produce a rhamnolipid-type biosurfactant. Here, we attempted to define the structural properties of this nat- ural product. The analysis of the extracted biosurfactant by thin-layer chromatography (TLC) revealed the presence of two compounds corresponding to those of authentic mono- and di-rhamnolipid. The identity of two structurally distinguished rhamnolipids was confirmed by 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy. Liquid chromatography/mass spectrometry (LC/MS) of extracted bio- surfactant revealed up to seventeen different rhamnolipid congeners. Further quantification showed di-rhamnolipids as the major compound (77.2%), while monorhamnolipids comprising a smaller pro- portion (22.8%) of MR01 biosurfactant. Rha-Rha-C 10 -C 10 was verified as the major component of the MR01 biosurfactant (35.93%). Cytotoxic activity of MR01 biosurfactant against human cancer Hela cells showed an excellent inhibitory effect of 5 g/ml. An isolated mutant strain (MR01-C) created by Gamma ray irradiation demonstrated more than one and a half-fold biosurfactant production and activity com- pared with the parent strain. Analysis of the biosurfactant produced by MR01-C showed the magnitude of di-rhamnolipids in the sample increased up to 88.6% (∼15% higher than control) and the quantity of Rha-Rha-C 10 -C 10 increased to 52.08% (∼45% higher than control). © 2010 Elsevier B.V. All rights reserved. 1. Introduction A variety of bacteria have been described that are abundant sources of useful bioactive secondary metabolites [1]. Among these compounds, biosurfactants are surface-active agents of great importance due to their unique biochemical properties, which are derived from their complex structures. The large interest in these natural compounds has arisen from their enormous potential in various industries. Examples of bacteria that are able to produce a large variety of these bioactive secondary metabolites are the Pseu- domonads. Pseudomonas aeruginosa is the most commonly isolated bacterium with rhamnolipid biosurfactant-producing capabilities [2]. These glycolipid-type biosurfactants have significant ten- sioactive and emulsifying properties with immense potential, particularly in soil/sand bioremediation and enhanced oil recov- ∗ Corresponding author. Tel.: +98 21 44580352; fax: +98 21 44580399. E-mail addresses: Akbari@nigeb.ac.ir, Kambizakb@yahoo.com (K.A. Noghabi). ery [3,4]. This has been the impetus behind most of the interest in the biochemical and structural characterization of this type of bio- surfactant. Rhamnolipid-type biosurfactants are categorized into two major clusters: the mono-rhamnolipids, which have one unit of rhamnose linked to one or two molecules of -hydroxyalkanoic acids and the di-rhamnolipids with two units of rhamnose simi- larly linked to -hydroxycarboxylic acids. It is well known that the production of rhamnolipid and its chemical composition is strain- specific and sensitive to culture conditions [5,6,7]. There are several reports that rhamnolipids produced by Pseudomonas sp. grown with different carbon sources can be mixtures of 4–28 different homologues, including where the monorhamnolipid Rh-C 10 C 10 is the predominant component [8–13]. Physical mutagenesis using different irradiation methodologies has been adopted as one of many strategies to mutate bacteria. A similar example was reported by Iqbal et al. [14], in which a gamma ray-induced mutant of P. aeruginosa S8 produced 2–3 times more biosurfactant than wild type cells. Raza et al. [15] also studied the ability of a gamma ray-induced Pseudomonas putida 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.06.026