Rhamnolipid Biosurfactant

SURFACTANTS

Surfactants are surface active agents and are defined as organic compounds that can enhance cleaning efficiency, emulsifying, wetting, dispersing, solvency, foaming/defoaming and lubricity of water-based compositions.  All surfactants share this chemical structure – a hydrophilic (water-loving) “head” and a hydrophobic (oil-loving) “tail” which is always a long (linear) chain of carbon atoms. Surfactants are generally classified into two groups:  petroleum chemistry-derived or synthetic surfactants, and living-cell produced biosurfactants.

The primary function of surfactants is to enhance the performance properties of water-based formulations including a range of ingredients such as other surfactants, solvents, thickeners, alkalis/salts, chelating agents, foamers/defoamers and fragrances. They are grouped by their ionic charge into four groups:  anionic, nonionic, cationic and amphoteric surfuctants.  Anionic surfactants are the largest group accounting for approximately 40% of world production;  they exhibit superior wetting and emulsifying properties and tend to be higher-foaming materials.

 

BIOSURFACTANTS

Biosurfactants are surface-active substances synthesised by living cells such as bacteria, fungi and yeast. Biosurfactants are generally non-toxic, environmentally benign and biodegradable. By comparison, chemical surfactants, which are usually derived from petroleum, are commonly toxic to health and ecosystems, and resist complete degradation. Biosurfactants have the properties of reducing the surface tension of a liquid, reducing the interfacial tension between two liquids, emulsifying/solubulizing water-insolubile or poor solubile substances, stabilising oil-in-water emulsions, demuslification and promoting foaming.  Interest in microbial surfactants has been steadily increasing in recent years due to their diversity, environmentally friendly nature, possibility of large-scale production, selectivity, performance under extreme conditions and potential applications in many industries such as pharmaceutics, cosmetics, food processing, agricultural production and in situ and ex-situ bioremediation.

 

RHAMNOLIPIDS

Rhamnolipids are members of the glycolipid biosurfactants and were discovered and first identified from Pseudomonas sp. in 1949 (Jarvis 1949). Since then, numerous producing microorganisms including bacteria, fungi and yeast have been reported to produce rhamnolipids. Pseudomonas aeruginosa species of soil bacteria have been identified as the most frequently isolated rhamnolipid producers (Nie, Yin et al. 2010).

An amphiphilic rhamnolipid molecule is composed of two moities. One half is the hydrophilic sugar part, mono- or di-rhamnose, and the hydrophobic lipid part possessing one or two 3-hydroxy fatty acid residues. These residues may either be both fully saturated or one may be saturated and the other unsaturated with either one or two double bonds. The lipid moiety is attached to the sugar by O-glycosidic linkage while the two 3-hydroxy acyl groups are joined together by the formation of an ester bond. The structure diversity of rhamnolipids is determined by the number of rhamnose (one or two) and fatty acid (one or two), and the fatty acid composition. The length of the constituent fatty acids and their combinations have been found to be largely variable. To date, over 40 different rhamnolipid congeners have been described in the scientific literature, though Rha-C10-C10 and Rha-Rha-C10-C10 are typically found to be the dominant components in a naturally occurring mixture. Rhamnolipids as members of glycolipids, are the most extensively studied biosurfactant. They are commonly classfied into two groups: monorhamnolipids and dirhamnolipids.

The functions of monorhamnolipids and dirhamnolipids include that of a natural surfactant, emulsifier, foaming/wetting agent, solubilizer, bactericide and fungicide, and anionic complexation agent.  Rhamnolipids may be manufactured in different forms such as raw or pure powder, pure paste-like/honey-like/syrup-like/wax-like and aqueous solution. Rhamnolipids are included in category IV of the EPA classification system, which includes nonirritant products (Haba 2003).

 

ANTIBACTERIAL ACTIVITY OF RHAMNOLIPIDS

Rhamnolipids have been shown active against many microorganisms including Gram-negative and Gram-positive bacteria, phytopathogenic fungi, algae, viruses as well as amoeba (Vatsa, Sanchez et al. 2010). It is known that a monorhamnolipid predominant mixture usually prepared to contain over 50 to 90% of Rha-C10-C10 is more active than a dirhamnolipid predominant mixture usually prepared to contain over 50 to 90% of Rha-Rha-C10-C10 (Samadi, Abadian et al. 2012). A comprehensive inventory of the microorganisms susceptible to rhamnolipids was well summarized by Vatsa and co-workers (Vatsa, Sanchez et al. 2010). The minimum inhibitory concentrations of the mixture of rhamnolipid  congeners were determined for some bacterial strains such as Staphylococcus aureus ATCC29213 (MIC = µg/ml), Methicillin-resistant Staphylococcus aureus ATCC33591 (MICs = 50 µg/ml) (Samadi, Abadian et al. 2012), Staphylococcus aureus ATCC6538 (MICs = 32 and 128 µg/ml) (Abalos 2001, Haba 2003), Staphylococcus epidermidis ATCC11228 (MICs = 32 and 8 µg/ml) (Abalos 2001, Haba 2003), Clostridium perfringens ATCC486 (MICs = 128 and 256 µg/ml) (Abalos 2001, Haba 2003). The discrepancy in the reported MICs for the same bacterial strains may be due to the variation of the purity and composition of rhamnolipid samples used and the quantification methods for ramnolipid samples.

 

ANTIMICROBIAL MECHANISMS OF ACTION OF RHAMNOLIPIDS

 The antimicrobial mechanisms of action of rhamnolipids are still not completely understood (Magalhaes 2013). The main mode of action of rhamnolipids against zoospore-producing phytopathogenic fungi has been better characterized to directly lyze zoospores via the interaction of rhamnolipids within plasma membranes of the zoospore (Vatsa, Sanchez et al. 2010). Bacterial cytoplasmic membranes are known to be the primary site of surfactant action (Nielsen, Kadavy et al. 2005). It was shown rhamnolipids are responsible for the membrane surface protein leakage due to alteration of the membrane permeability by possible aggregation and formation of transmembrane pores channeling to the periplasm (King 1991, Sotirova, Spasova et al. 2008). Rhamnolipids were capable to cause the changes of cell surface hydrophobicity and loss of lipopolysaccharide (LPS) from the outer membrane (Al-Tahhan, Sandrin et al. 2000). Bacteria with smooth-form LPS are resistant to the action of hydrophobic antibiotics. In contrast, bacteria without LPS are more susceptible to the action of hydrophobic antibiotics. Rhamnolipids were recently demonstrated to remarkably increase the negatively charged phospholipid cardiolipin in the presence of specific thiosulfonic alkyl esters (Sotirova, Avramova et al. 2012). The changes in phospholipid’s composition of bacterial membrane may lead to increased susceptibility of bacteria to some antibiotics. Mass spectrometric study of rhamnolipid biosurfactant suggested that the formation of stable supramolecule complexes of rhamnolipids with membrane phospholipids is a possible molecular mechanism of their antimicrobial activity (Samadi, Abadian et al. 2012).

 

PRODUCTION AND PURIFICATION OF RHAMNOLIPIDS

Production and purification of rhamnolipid samples from the fermentation broths were according to publicly available standard protocols and optimized for column purification, TLC preparation and HPLC purification, LC-MS and MS/MS analyses to achieve and obtain the desired purities of rhamnolipids (Ochsner, Reiser et al. 1995, Deziel, Lepine et al. 1999, Wang, Fang et al. 2007, Lotfabad, Abassi et al. 2010, Nitschke, Costa et al. 2010, Zgola-Grzeskowiak and Kaczorek 2011)

Rhamnolipids naturally occur as a mixture of dirhamnolipid and monorhamnolipid congeners regardless of microbial producers and fermentation media. Typically, dirhamnolipids and monorhamnolipids with a fatty acid chain length of 10 carbons (Rha-Rha-C10-C10 and Rha-C10-C10) are predominant in the naturally occurring mixture. After preparation by repeated acidic precipitation and solvent extraction, followed by multiple steps of column chromatography purification, different purity levels of rhamnolipids can be obtained. Isolation of a single congener from the mixture of rhamolipids is technically and practically achievable but it is not cost effective. For this reason, practices in antimicrobial activity tests reported in literature have been commonly or unexceptionally conducted with a mixture of rhamnolipid congeners which were purified from fermentation broth and comprised of Rha-Rha-C10-C10 or Rha-C10-C10 or both as predominant congeners as well as minor or trace amounts of other rhamnolipid congeners. LC-MS and MS/MS analysis can be used to readily detect the distinct rhamnolipid congeners in a fractionated and relatively pure sample preparation (Samadi, Abadian et al. 2012). The purity level of rhamnolipid preparation is critically important for accurate quantification, dosage and maximal elimination of any potential interference caused by the contamination of impure substances such as spent medium  and an array of uncharacterized bacterial metabolites. For pharmaceutical, cosmetic and food additive uses, the high purity and quality of rhamnolipids (purity level from 90% to 99%) are required to meet regulatory standards.

 

DETERMINATION OF PURITY, CONCENTRATION AND COMPOSITION OF RHAMNOLIPIDS

 The purity and concentration of a rhamnolipid sample can be determined by TLC or TLC and HPLC using the highly pure (95%) rhamnolipid product (commercially available from Sigma-Aldrich). The compositions of a rhamnolipid sample can be determined by LC-MS or LC-MS plus MS/MS analyses (Ochsner, Reiser et al. 1995, Deziel, Lepine et al. 1999, Wang, Fang et al. 2007, Lotfabad, Abassi et al. 2010, Nitschke, Costa et al. 2010, Zgola-Grzeskowiak and Kaczorek 2011).

 

For information about the various industrial applications of rhamnolipids, click here.

 

Literature Cited

Abalos, A., Pinazo, A., Infante, M.R., Casals, M., Garcia, F., Manresa, A. (2001). “Physicochemical and Antimicrobial Properties of New Rhamnolipids Produced by Pseudomonas aeruginosa AT10 from Soybean Oil Refinery Wastes.” Langmuir 17: 1367-1371.

Al-Tahhan, R. A., T. R. Sandrin, A. A. Bodour and R. M. Maier (2000). “Rhamnolipid-induced removal of lipopolysaccharide from Pseudomonas aeruginosa: effect on cell surface properties and interaction with hydrophobic substrates.” Appl Environ Microbiol 66(8): 3262-3268.

Deziel, E., F. Lepine, D. Dennie, D. Boismenu, O. A. Mamer and R. Villemur (1999). “Liquid chromatography/mass spectrometry analysis of mixtures of rhamnolipids produced by Pseudomonas aeruginosa strain 57RP grown on mannitol or naphthalene.” Biochim Biophys Acta 1440(2-3): 244-252.

Haba, E., Pinazo, A., Jauregui, O., Espuny, M.J., Infante, M.R., Manresa, A. (2003). “Physicochemical Characterization and Antimicrobial Properties of Rhamnolipids Produced by Pseudomonas aeruginosa 47T2 NCBIM 40044.” Biotechnol. Bioeng. 81(3): 316-322.

Jarvis, F., Johnson, M.A. (1949). “A glycolipid produced by Pseudomonas aeruginosa.” J. Am. Chem. Soc.(71).

Jiang, L., Long, X., Meng, Q. (2013). “Rhamnolipids enhance epithelial permeability in Caco-2 monolayers.” Int. J. Pharm. 446(1-2):130-5.

King, A. T., Davey, M.R., Mellor, I.R., Muligan, B.J., Lowe, K.C. (1991). “Surfactant effects on yeast cells.” Enzyme Microb Technol 13.: 148-153.

Lotfabad, T. B., H. Abassi, R. Ahmadkhaniha, R. Roostaazad, F. Masoomi, H. S. Zahiri, G. Ahmadian, H. Vali and K. A. Noghabi (2010). “Structural characterization of a rhamnolipid-type biosurfactant produced by Pseudomonas aeruginosa MR01: enhancement of di-rhamnolipid proportion using gamma irradiation.” Colloids Surf B Biointerfaces 81(2): 397-405.

Magalhaes, L., Nitschke, M. (2013). “Antimicrobial activity of rhamnolipids against Listeria monocytogenes and their synergistic interaction with nisin.” Food Control 29: 138-142.

Maier, R. M. and G. Soberon-Chavez (2000). “Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications.” Appl Microbiol Biotechnol 54(5): 625-633.

Nie, M., X. Yin, C. Ren, Y. Wang, F. Xu and Q. Shen (2010). “Novel rhamnolipid biosurfactants produced by a polycyclic aromatic hydrocarbon-degrading bacterium Pseudomonas aeruginosa strain NY3.” Biotechnol Adv 28(5): 635-643.

Nielsen, L. E., D. R. Kadavy, S. Rajagopal, R. Drijber and K. W. Nickerson (2005). “Survey of extreme solvent tolerance in gram-positive cocci: membrane fatty acid changes in Staphylococcus haemolyticus grown in toluene.” Appl Environ Microbiol 71(9): 5171-5176.

Nitschke, M., S. G. Costa and J. Contiero (2010). “Structure and applications of a rhamnolipid surfactant produced in soybean oil waste.” Appl Biochem Biotechnol 160(7): 2066-2074.

Ochsner, U. A., J. Reiser, A. Fiechter and B. Witholt (1995). “Production of Pseudomonas aeruginosa Rhamnolipid Biosurfactants in Heterologous Hosts.” Appl Environ Microbiol 61(9): 3503-3506.

Piljac, A., T. Stipcevic, J. Piljac-Zegarac and G. Piljac (2008). “Successful treatment of chronic decubitus ulcer with 0.1% dirhamnolipid ointment.” J Cutan Med Surg 12(3): 142-146.

Samadi, N., N. Abadian, R. Ahmadkhaniha, F. Amini, D. Dalili, N. Rastkari, E. Safaripour and F. A. Mohseni (2012). “Structural characterization and surface activities of biogenic rhamnolipid surfactants from Pseudomonas aeruginosa isolate MN1 and synergistic effects against methicillin-resistant Staphylococcus aureus.” Folia Microbiol (Praha) 57(6): 501-508.

Sotirova, A., T. Avramova, S. Stoitsova, I. Lazarkevich, V. Lubenets, E. Karpenko and D. Galabova (2012). “The importance of rhamnolipid-biosurfactant-induced changes in bacterial membrane lipids of Bacillus subtilis for the antimicrobial activity of thiosulfonates.” Curr Microbiol 65(5): 534-541.

Sotirova, A. V., D. I. Spasova, D. N. Galabova, E. Karpenko and A. Shulga (2008). “Rhamnolipid-biosurfactant permeabilizing effects on gram-positive and gram-negative bacterial strains.” Curr Microbiol 56(6): 639-644.

Vatsa, P., L. Sanchez, C. Clement, F. Baillieul and S. Dorey (2010). “Rhamnolipid Biosurfactants as New Players in Animal and Plant Defense against Microbes.” Int J Mol Sci 11(12): 5095-5108.

Wang, Q., X. Fang, B. Bai, X. Liang, P. J. Shuler, W. A. Goddard, 3rd and Y. Tang (2007). “Engineering bacteria for production of rhamnolipid as an agent for enhanced oil recovery.” Biotechnol Bioeng 98(4): 842-853.

Zgola-Grzeskowiak, A. and E. Kaczorek (2011). “Isolation, preconcentration and determination of rhamnolipids in aqueous samples by dispersive liquid-liquid microextraction and liquid chromatography with tandem mass spectrometry.” Talanta 83(3): 744-750.

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