Langmuir 2001, 17, 1367-1371
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Physicochemical and Antimicrobial Properties of New Rhamnolipids Produced by Pseudomonas aeruginosa AT10 from Soybean Oil Refinery Wastes A. Abalos,† A. Pinazo,‡ M. R. Infante,‡ M. Casals,§ F. Garcı´a,| and A. Manresa*,† Departament de Microbiologia i Parasitologia Sanita` ries, Facultat de Farma` cia, Universitat de Barcelona, E-08028 Barcelona, Spain, Department of Surfactant Technology, CID (CSIC), Barcelona, Spain, Departamento de Quı´mica, Facultad de Ciencias Naturales y Matema´ ticas, Universidad de Oriente, Lumumba s/n, 90500 Santiago de Cuba, Cuba, and Laboratori Sanitat Vegetal, DARP, Barcelona, Spain Received August 14, 2000. In Final Form: December 6, 2000 The rhamnolipids produced by Pseudomonas aeruginosa strains are often a mixture of several homologues. Up to seven (R2C10C10 + R1C10C10 + R2C10C12 + R1C10C12 + R1C12:1C10 + R1C12:2 + R1C8:2 ) have been identified in cultures of P. aeruginosa AT10 from soybean oil refinery wastes. This study deals with the production, purification, and identification of rhamnolipids in the mixture (M7), as well as their physical and chemical characterization and the evaluation of their antimicrobial properties. The surface tension decreases to 26.8 mN/m and the critical micelle concentration value to 1.2 × 102 mg/L. These molecules show excellent antifungal properties against Aspergillus niger and Gliocadium virens (16 µg/mL) and C. globosum, P. crysogenum, and A. pullulans (32 µg/mL), whereas the growth of the phytopathogenic fungi B. Cinerea and R. solani was inhibited at 18 µg/mL.
Introduction Biosurfactants are valuable microbial amphiphilic molecules with effective surface-active and biological properties applicable to several industries and processes.1 They are synthesized by microbes, especially during their growth on water-immiscible substrates,2 which are an alternative to chemically prepared conventional surfactants. Because of their structural diversity (i.e., glycolipids, lipopeptides, fatty acids, etc.),3-6 low toxicity, and biodegradability, these molecules could be widely used in cosmetic, pharmaceutical, and food processes as emulsifiers, humectants, preservatives, and detergents. Moreover, they are ecologically safe and they can be applied in bioremediation and waste treatments.7 Biosurfactants favor the uptake of hydrophobic substrates into the cell. They can be produced by various substrates,8 mainly * To whom correspondence should be sent at the Laboratorio de Microbiologı´a, Facultad de Farmacia, Universidad de Barcelona, Joan XXXIII s/n, 08028 Barcelona. Espan˜a. E-mail: manresa@ farmacia.far.ub.es. † Universitat de Barcelona. ‡ Department of Surfactant Technology, CID (CSIC). § Universidad de Oriente. | Laboratori Sanitat Vegetal, DARP. (1) Cooper, D. G.; Zajic, J. E. Adv. Appl. Microbiol. 1980, 26, 229253. (2) Ochsner, U.; Hembach, T.; Fiechter, A. Adv. Biochem. Eng. Biotechnol. 1995, 53, 89-118. (3) Babu, P. S.; Vaidya, A. N.; Bal, A. S.; Kapur, R.; Juwarkar, A. Khanna, P.; Biotechnol. Lett. 1996, 18, 263-268. (4) Mercade´, M. E.; Espuny, M. J.; Manresa, A. Recent Res. Dev. Oil Chem. 1997, 1, 177-185. (5) Haba, E.; Espuny, M. J.; Busquets, M.; Manresa, A. J. Appl. Microbiol. 2000, 88, 379-387. (6) Daniel, H. J.; Otto, R. T.; Reuss, M.; Syldatk, C. Appl. Microbiol. Biotechnol. 1999, 51, 40-45. (7) Banat, I.; Makkar, R.; Cameotra, S. Appl. Microbiol. Biotechnol. 2000, 53, 495-508. (8) Volbrecht, E.; Heckmann, R.; Wray, V.; Nimtz, M.; Lang, S. Appl. Microbiol. Biotechnol. 1998, 50, 530-537.
Figure 1. General structure of rhamnolipid: n ) 4, 6, 8 for length chain C8, C10, or C12 carbon atoms. In dirhamnolipids, R2 is L-rhamnosyl. R1 is H or 3-hydroxydecanoate.
renewable resources such as vegetable oils,9 which are economical but have been scarcely reported. The biosurfactants produced by Pseudomonas aeruginosa strains, or rhamnolipids (rhamnnose-containing glycolipids) (Figure 1), are often a mixture of various homologues. They have traditionally been reported as mixtures of two or four species: GL-A and GL-B10 or RL1, RL2, RL3, and RL4.11,12 Recently, a wide variety of rhamnolipid homologues has been described.13 Up to seven homologues have been identified in cultures of Pseudomonas aeruginosa AT10, the two main compounds being L-rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate (R1C10C10), referred to as RL1, and L-rhamnosylrhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate (R2C10C10), as RL2.12 This study deals with (a) the production, purification, and identification of a new rhamnolipid homologue mixture produced by Pseudomonas aeruginosa AT10 from a soybean oil refinery waste, (b) the physical and chemical characterization of the mixtures M7 (R2(9) Desai. J. D. Microbiol. Mol. Biol. Rev. 1997, 61, 47-64. (10) Itoh, S.; Honda, H.; Tomita, F.; Suzuki, T. J. Antibiot. 1971, 24, 855-859. (11) Syldatk, C.; Lang, S.; Matulovic, V.; Wagner, F. Z. Naturforsch. 1985, 40c, 61-67. (12) Lang, S.; Wullbrandt, D. Appl. Microbiol. Biotechnol. 1999, 51, 22-32. (13) De´ziel, E.; Le´pine, F.; Dennie, D.; Boismenu, D.; Mamer, O.; Villemur, R. Biochim. Biophys. Acta 1999, 1440, 244-252.
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C10C10 + R1C10C10 + R2C10C12 + R1C10C12 + R1C12:1C10 + R1C12:2 + R1C8:2 ) and M6 (R1C10C10 + R2C10C12 + R1C10C12 + R1C12:1C10 + R1C12:2 + R1C8:2) and R2C10C10, and (c) the evaluation of their antimicrobial properties. We aimed to show the applicability of the whole rhamnolipid mixture as a new surface-active and antimicrobial product in several technological processes and formulations. Materials and Methods Strain and Culture Conditions. Pseudomonas aeruginosa AT10 was isolated from contaminated soils of the ERASOL foodoil refinery in Santiago de Cuba (Cuba). Experiments were carried out in 250-mL baffled Erlenmeyer flasks containing 50 mL of medium, whose composition was as follows (g L-1): NaNO3, 5; K2HPO4/KH2PO4, 4; FeSO4‚7H2O, 0.011; KCl, 0.1; MgSO4‚7H2O, 0.5; CaCl2, 0.01; yeast extract, 0.01, and 0.05 mL of a trace-element solution. Waste free fatty acids (WFFAs) (5%) supplied by ERASOL food-oil refinery in Santiago de Cuba (Cuba) was added as carbon substrate. The medium was sterilized for 20 min at 120 °C and 1 atm. Cultures were incubated in a reciprocal shaker (150 rpm) at 30 °C. Analytical Methods. The WFFAs used as carbon substrate were analyzed on a Hewlett-Packard 5890 series II chromatograph (Hewlett-Packard, Palo Alto, CA) equipped with splitless injector and flame ionization detector (FID). A Supelco 2380, 60 m × 0.25 mm i.d. fused silica capillary column was used at a temperature of 200 °C (isothermal) and a helium flow rate of 0.57 mL/min. The split ratio was 1:100, and peaks were integrated on a HP-Chem integrator (Hewlett-Packard). Rhamnolipids were quantified as rhamnose content by a colorimetric method by using a standard of rhamnose.14 The rhamnolipid content was calculated by multiplying the rhamnose concentration by 3.0, which represents the rhamnolipid/rhamnose correlation.10 Cell growth was monitored by measuring the protein content of the culture following the method of Lowry.15 Rhamnolipid Recovery. Rhamnolipids were harvested for 96 h and recovered from the supernatant after removal of cells by centrifugation at 5500g for 10 min in a Centrikon T-124 centrifuge (Kontron, Milano, Italy). They were then purified following a modification of the Reiling method.16 The supernatant was acidified to pH 6.1 and then passed through an Amberlite XAD-2 adsorption chromatography column (Sigma, St. Louis, MO). Rhamnolipid Isolation and Identification. Individual rhamnolipids were separated and identified by high-performance liquid chromatography (HPLC) coupled to a mass spectrometer (MS) using a Waters 2690 separation module (Waters, Midford, MA). The samples (10 µL) were analyzed by HPLC using a HYPERSIL C8 WP-300, 150 × 4.6 mm column (Teknochroma, San Cugat, Spain). An acetonitrile-water gradient was used starting with 70% acetonitrile for 4 min, followed by 70-100% acetonitrile for 40 min. The HPLC flow rate was 1 mL/min. In an HPLC-electrospray (ES)-MS, postcolumn addition of acetone at 200 µL/min was carried out using a Phoenix 20 (Carlo Erba) syringe pump. Mobile phase and acetone were mixed in a tee (Valco), and a split system (1/50) was used to introduce the effluent into the ES. MS was performed using a VG Platform II (Micromass, Manchester, U.K.) quadrupole mass spectrometer equipped with a pneumatically assisted electrospray (ES) ion source. Negative ion mode was used. Full scan data were obtained by scanning from m/z 200 to 800 in centroid mode using a cycle time of 1 s and an interscan time of 0.1 s. The working conditions for the ES were as follows: drying nitrogen was heated at 80 °C and introduced into the capillary region at a flow rate of 400 L/h. The capillary was held at a potential of -3.5 kV. The extraction voltage was - 35 V. (14) Chandrasekaran, E. V.; Bemiller, J. N. Constituent analysis of glucosamonoglucans. In Methods Carbohydrate Chemistry; Wrhiste, L, Wolfrom, M. L., Eds; Academic Press: New York, 1980; Vol. III, pp 89-97. (15) Lowry, O. H.; Rosebrought, N. J.; Farr, A.; Randall, R. J. J. Biol. Chem. 1951, 139, 265-274. (16) Reiling, H. E.; Thanei-wyss, U.; Guerra-Santos, L. H.; Hirt, R.; Ka¨ppeli, O.; Fiechter, A. Appl. Environ. Microbiol. 1986, 51, 985-989.
Abalos et al. Physicochemical Characterization. Equilibrium surface tension was measured by the Wilhelmy plate technique with a Kru¨ss K12 tensiometer. The instrument had been calibrated against Milli-Q-4 ultrapure distilled water (Millipore). Solutions with various concentrations of surfactants were obtained by successive dilutions of a concentrated sample (2 × 103 mg L-1) prepared by weight in Millipore ultrapure water. To reach the equilibrium, all sample solutions had been aged in appropriate cells at room temperature (25 °C). The platinum plate and all the glassware used were cleaned in a chromic mixture. Assay of Antimicrobial Activity. Antimicrobial activities were determined according to MIC values, defined as the lowest concentration of antimicrobial agent that inhibits the development of visible growth after 24 h of incubation at 37 °C. Antibacterial activity was monitored using a 2-fold serial rhamnolipid dilution technique after incubation for 24 h at 37 °C.17 Antimicrobial activity against yeast and fungi was measured in solid medium Sabouraud agar plates incubated for 72 h at 25 °C.18 A wide range of Gram-positive and Gram-negative bacteria were tested: Alcaligenes faecalis ATCC 8750, Bordetella bronchiseptica ATCC 4617, Citrobacter freundii ATCC 22636, Enterobacter aerogenes CECT 689, Escherichia coli ATCC 8739, Klebsiella pneumoniae var. pneumoniae CECT 178, Proteus miravillis CECT 170, Pseudomonas aeruginosa ATCC 9027, Salmonella thyphimurium ATCC 16028, Serratia marcescens CECT 274, Bacillus subtilis ATCC 6633, Bacillus cereus var. mycoide ATCC 11778, Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 11228, Streptoccocus faecalis ATCC 10541, Micrococcus luteus ATCC 9631, Arthrobacter oxidans ATCC 8010, Mycobacterium phlei ATCC 41423, and Clostridium perfringes ATCC 486. Yeast: Candida albicans ATCC 10231, Rhodotorula rubra CECT 1158, Saccharomyces cerevisiae ATCC 9763. Fungi strains: Aspergillus niger ATCC 14604, Aureobasidium pullulans ATCC 9348, Chaetonium globosum ATCC 6205, Gliocadium virens ATCC 4645, Penicillium chrysogeum CECT 2802, Penicillium funiculosum CECE 2914, Colletotrichum gloesporioides, Botrytis cinerea, Rhizotecnia solani, and Fusarium solani.
Results and Discussion Rhamnolipid Production. Pseudomonas aeruginosa AT10 was incubated in a mineral salts medium with residual free fatty acid as carbon source. Biomass was measured as the protein content of the whole culture, as shown in Figure 2. Cell growth reached 6.5 g/L of proteins after 17 h of incubation, when rhamnolipid accumulation was detected (0.2 g/L) and the surface tension was 39.7 mN/m, after which it decreased to 32 mN/m (24 h of incubation). Rhamnolipids emulsified the carbon substrate, thus favoring the uptake inside the cell. The final production of rhamnolipids, 9.5 g/L, was reached in two main stages. During the first, 3.54 g/L of rhamnolipid was produced with a volumetric productivity of 0.06 g of rhamnolipids/(L h). During the second (72-96 h), the concentration increased by 0.22 g/(L h) and most of the product was secreted to the medium (5.64 g/L). The fatty acid analysis of the substrate revealed the following (w/w): 17%, the most volatile fraction (256 >256 32 128 16 >256 >256
Gram positives Arthrobacter oxidans Bacillus cereus v. mycoide Bacillus subtillis Microccocus luteus Mycobacterium phlei Staphyloccocus aureus Staphyloccocus epidermidis Streptoccocus faecalis Clostridium perfringes
16 64 64 32 16 128 8 64 256
Yeasts Candida albicans Sacharomyces cereviciae Rhodotorula rubra
>256 >256 >256
Fungi Aerobacidium pullulants Aspergillus niger Chaetonium globusum Gliocadium virens Penicillium crysogenum Penicillium funiculosum Botrytis cinerea Colletotrichum gloesporioides Rhizotecnia solani
32 16 32 16 32 128 18 65 18
Regarding Gram-positive bacteria, M7 inhibits Bacillus at 64 µg/mL, in contrast with the R2C10C10 (RL1) and R1C10 (RL3) mixture, which prevented growth of Bacillus at 35 µg/mL, as shown by Lang and Wagner.26 Vollbrecht et al.27 reported an antimicrobial effect of new oligosaccharide lipids produced by Tsukamurella (about 50 µg/mL) against Bacillus megaterium. Sophorose lipids also prevent the growth of S. epidermidis (8 µ/mL)26 and Mycobacterium phlei (16 µg/mL). No antimicrobial activity was observed on the yeast strains tested, as shown Table 3. The antifungal activity of rhamnolipids from Pseudomonas AT10 on filamentous fungi is presented in Table 3. The MIC values against Aspergillus niger, Gliocadium virens (16 µg/mL), C. globosum, P. crysogenum, and A. pullulans (32 µg/mL) were excellent. The inhibitory activity against phytopathogen fungi was assayed. Growth of B. cinerea and R. solani was inhibited at 18 µg/mL, that of C. gloesporioides and F. solani at 65 µg/mL, but that of P. funiculosum at 128 µg/mL. Few data on the antifungal activity of biosurfactants are available. Mannosylerythritol lipids (MEL-A and MEL-B) from Candida antarctica show weak activity against Aspergillus niger.28 Vollbrecht et al.27 have reported antimicrobial activity of oligosaccharide lipids below 100 µg/mL. The target of the chemical surfactant is the cell envelope. Therefore, the physicochemical properties of biosurfactants are responsible for the effect on the cell surface, as well as the structure of microbial cells. Although the mechanism of action of the anionic surfactants on the cell membranes and proteins has been extensively studied, (28) Kitamoto, D.; Yanagishita, H.; Shinbo, T.; Kamisawa, C.; Nakane, T.; Nakahara, T. J. Biotechnol. 1993, 29, 91-96.
Properties of Rhamnolipids
the different effect on Gram-positive and Gram-negative bacteria29 has not been elucidated. The rhamnolipids produced by Pseudomonas aeruginosa AT10 are a mixture of seven homologues. Due to its physicochemical properties and high antimicrobial activity, it can be regarded as a useful tool in bioremediation processes, cosmetics, and food industrial applications. (29) Fredell, D. Cationic Surfactants Analytical and biological evaluation. In Biological properties and applications of cationic surfactants; Marcel Dekker: New York, 1994; Vol. 53, pp 30-60.
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Acknowledgment. We thank the fellowship of the Agencia Espan˜ola de Cooperacio´n con Iberoame´rica (AECI), and the project AMB 96-1429 from the CICYT 199956R 00024. We are also grateful to the Technical services of the University of Barcelona for the HPLC-MS analysis. Note Added after ASAP Posting. This article was released ASAP on 2/10/2001 with an error in the author byline. The corrected version was posted 2/27/2001. LA0011735
