Production and characterization of rhamnolipids from Pseudomonas aeruginosa san ai

UDC Pseudomonas aeruginosa san-ai: 577.115.002.2 Original scientific paper J. Serb. Chem. Soc. 77 (1) 27–42 (2012) JSCS–4246 Production and characterization of rhamnolipids from Pseudomonas aeruginosa san-ai MILENA G. RIKALOVIĆ1, GORDANA GOJGIĆ-CVIJOVIĆ2, MIROSLAV M. VRVIĆ1,2 and IVANKA KARADŽIĆ3* 1Faculty of Chemistry, University of Belgrade, Studentski trg 12–16, Belgrade, Serbia, 2Department of Chemistry, ICTM, University of Belgrade, Njegoševa 12, Belgrade, Serbia and 3School of Medicine, Department of Chemistry, University of Belgrade, Višegradska 26, Belgrade, Serbia (Received 11 February, revised 3 August 2011) Abstract: The production and characteristics of rhamnolipid biosurfactant obtained by the strain Pseudomonas aeruginosa san-ai were investigated. With regard to the carbon and nitrogen sources, several media were tested to enhance the production of rhamnolipids. Phosphate-limited proteose peptone–ammonium salt (PPAS) medium supplemented with sunflower oil as a source of carbon and mineral ammonium chloride and peptone as nitrogen sources greatly improved the production of rhamnolipid, from 0.15 on basic PPAS (C/N ratio 4.0) to 3 g L-1 on optimized PPAS medium (C/N ratio 7.7). Response surface methodology analysis was used for testing the effect of three factors, i.e., temperature, concentration of carbon and nitrogen source (mass %), in the optimized PPAS medium on the production of rhamnolipid. The isolated rhamnolipids were characterized by infrared (IR) spectroscopy and electrospray ionization mass spectrometry (ESI–MS). The IR spectra confirmed that the isolated compound corresponded to the rhamnolipid structure, whereas MS indicated that the isolated preparation was a mixture of mono-rhamno-mono-lipidic, mono-rhamno-di-lipidic and di-rhamno-di-lipidic congeners. Keywords: rhamnolipids; Pseudomonas aeruginosa; renewable sources. INTRODUCTION Biosurfactants are microbial secondary metabolites that appear to play a role whenever a microbe encounters an interface.1 Biosurfactants are important for motility, cell–cell interactions (biofilm formation, maintenance and maturation, quorum sensing, amensalism and pathogenicity) and cellular differentiation, substrate accession (via direct interfacial contact and pseudosolubilization of substrates), as well as avoidance of toxic elements and compounds. They may also * Corresponding author. E-mail: doi: 10.2298/JSC110211156R 27 Available online at www.shd.org.rs/JSCS/ ________________________________________________________________________________________________________________________ 2012 Copyright (CC) SCS 28 RIKALOVIĆ et al. be used as carbon and energy storage molecules, as a protective mechanism against high ionic strength, and may simply be byproducts released in response to environmental changes (e.g., extracellular coverings).2 Almost all surfactants currently in use are chemically derived from petroleum. However, biosurfactants have several advantages over the chemical surfactants, such as lower toxicity, higher biodegradability, better environmental compatibility, higher foaming, high selectivity and specific activity at extreme temperatures, pH and salinity, and the ability to be synthesized from renewable feedstock.1 Due to these properties, biosurfactants are becoming important biotechnology products for industrial and medical applications.3 They can be used as emulsifiers, de-emulsifiers, wetting and foaming agents, functional food ingredients and as detergents in petroleum, petrochemicals, environmental management, agrochemicals, foods and beverages, cosmetics and pharmaceuticals, and in the mining and metallurgical industries. Surfactants also play an important role in enhanced oil recovery by increasing the apparent solubility of petroleum components and effectively reducing the interfacial tensions of oil and water in situ.4 The main factor limiting commercialization of biosurfactants is associated with their non-economic large-scale production. To overcome this obstacle and to compete with synthetic surfactants, an inexpensive substrate and effective microorganism have to be intensively developed for biosurfactant production. Agroindustrial wastes are considered as promising substrates for biosurfactant production, which could alleviate many processing industrial waste management problems.5 The fact should be noted that although the literature mentions a number of microbe producers with potential to be advantageous for increasing production and efficiency, in practice, this has only been confirmed for a few genera such as Bacillus, Candida and Pseudomonas.1 Regardless of these problems, the production of microbial surfactants follows the trend of green chemistry and forms the basis of modern industrial processes. The creation of an ecological society, which is in harmony with its surroundings, is now, with green chemistry, the greatest challenge for science and mankind. Biosurfactants can be divided into two classes: low-molecular-mass molecules, which efficiently lower surface and interfacial tension, and high-molecular-mass polymers, which are more effective as emulsion stabilizing agents. The classes of low-mass surfactants include glycolipids, lipopeptides and phospholipids, whereas high-mass ones include polymeric and particulate surfactants. Most biosurfactants are either anionic or nonionic and the hydrophobic moiety is based on long-chain fatty acids or their derivatives whereas the hydrophilic portion can be a carbohydrate, amino acid, phosphate or cyclic peptide. Bacteria are the predominant group of surfactant-producing organisms.3 Pseudomonas species synthesize both classes of surfactants, low- and high-molecular-mass molecules, but are commonly mentioned as rhamnolipid (RL) producers.2,6 Available online at www.shd.org.rs/JSCS/ ________________________________________________________________________________________________________________________ 2012 Copyright (CC) SCS RHAMNOLIPIDS FROM Pseudomonas aeruginosa SAN-AI 29 Rhamnolipids (RLs) belong to class of low-molecular-mass molecules. The principal rhamnolipids: mono-rhamno-di-lipidic congener and di-rhamno-di-lipidic congener, consist of one or two L-rhamnose units and two units of β-hydroxydecanoic acid (RL1 and RL2 in Fig. 1), while mono-rhamno-mono-lipidic congener and di-rhamno-mono-lipidic congener, consisting of one or two L-rhamnose and one unit of β-hydroxydecanoic acid, are biosynthesized only under certain cultivation conditions (RL3 and RL 4 in Fig. 1).7 Rhamnolipids are secondary metabolites, and as such, their production coincides with the onset of the stationary phase of microbial growth.8 Rhamnolipid production seems possible from most carbon sources supporting bacterial growth. Nevertheless, oil of vegetable origin, such as soybean, corn, canola, and olive, provides the highest productivity. Elevated C/N and C/P ratios promote the production of rhamnolipids, while high concentrations of d (...truncated)