Rhamnolipid-induced removal of lipopolysaccharide from Pseudomonas aeruginosa: effect on cell surface properties and interaction with hydrophobic substrates

Abstract

Little is known about the interaction of biosurfactants with bacterial cells. Recent work in the area of biodegradation suggests that there are two mechanisms by which biosurfactants enhance the biodegradation of slightly soluble organic compounds. First, biosurfactants can solubilize hydrophobic compounds within micelle structures, effectively increasing the apparent aqueous solubility of the organic compound and its availability for uptake by a cell. Second, biosurfactants can cause the cell surface to become more hydrophobic, thereby increasing the association of the cell with the slightly soluble substrate. Since the second mechanism requires very low levels of added biosurfactant, it is the more intriguing of the two mechanisms from the perspective of enhancing the biodegradation process. This is because, in practical terms, addition of low levels of biosurfactants will be more cost-effective for bioremediation. To successfully optimize the use of biosurfactants in the bioremediation process, their effect on cell surfaces must be understood. We report here that rhamnolipid biosurfactant causes the cell surface of Pseudomonas spp. to become hydrophobic through release of lipopolysaccharide (LPS). In this study, two Pseudomonas aeruginosa strains were grown on glucose and hexadecane to investigate the chemical and structural changes that occur in the presence of a rhamnolipid biosurfactant. Results showed that rhamnolipids caused an overall loss in cellular fatty acid content. Loss of fatty acids was due to release of LPS from the outer membrane, as demonstrated by 2-keto-3-deoxyoctonic acid and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and further confirmed by scanning electron microscopy. The amount of LPS loss was found to be dependent on rhamnolipid concentration, but significant loss occurred even at concentrations less than the critical micelle concentration. We conclude that rhamnolipid-induced LPS release is the probable mechanism of enhanced cell surface hydrophobicity.

FIG. 1

Growth, KDO, and cell surface hydrophobicity of P. aeruginosa ATCC 27853 grown on 1% glucose in the presence or absence of rhamnolipid. ○, no rhamnolipid; ●, 6 mM rhamnolipid. Each point represents the average and standard deviation of three replicate samples.

FIG. 2

Growth, KDO, and cell surface hydrophobicity of P. aeruginosa ATCC 9027 grown on 1% glucose in the presence or absence of rhamnolipid. ○, no rhamnolipid; ●, 6 mM rhamnolipid. Each point represents the average and standard deviation of three replicate samples.

FIG. 3

Growth, KDO, and cell surface hydrophobicity of P. aeruginosa ATCC 27853 grown on 1% hexadecane in the presence or absence of rhamnolipid. ○, no rhamnolipid; ●, 6 mM rhamnolipid. Each point represents the average and standard deviation of three replicate samples.

FIG. 4

Growth, KDO, and cell surface hydrophobicity of P. aeruginosa ATCC 9027 grown on 1% hexadecane in the presence or absence of rhamnolipid. ○, no rhamnolipid; ●, 6 mM rhamnolipid. Each point represents the average and standard deviation of three replicate samples.

FIG. 5

Effect of rhamnolipid on LPS released from cell suspensions of P. aeruginosa ATCC 27853. Cell suspensions were adjusted to an OD of 1.0 and were incubated with the respective rhamnolipid concentration for 8 h, and supernatant samples were assayed for LPS. Each point represents the average and standard deviation of three replicate samples.

FIG. 6

Effect of rhamnolipid on LPS released from cell suspensions of P. aeruginosa ATCC 9027. Cell suspensions were adjusted to an OD of 1.0 and were incubated with the respective concentration of rhamnolipid for 8 h, and supernatant samples were assayed for LPS. Each point represents the average and standard deviation of three replicate samples.

FIG. 7

A silver-stained SDS-PAGE of concentrated (10×) supernatants of suspensions of P. aeruginosa ATCC 27853. Lane 1, buffer; lane 2, 300 μg of rhamnolipid; lane 3, 5 μg of bovine serum albumin; lane 4, supernatant of P. aeruginosa ATCC 27853 treated only with MSM for 8 h; lane 5, supernatant of P. aeruginosa ATCC 27853 treated with 6 mM rhamnolipid for 8 h; lane 6, supernatant of P. aeruginosa ATCC 27853 treated only with MSM for 24 h; lane 7, supernatant of P. aeruginosa ATCC 27853 treated with 6 mM rhamnolipid for 24 h; lane 8, 5 μg of P. aeruginosa serotype 10 LPS; lane 9, 50 μg of P. aeruginosa serotype 10 LPS; lane 10, 1 μg of P. aeruginosa serotype 10 LPS.

FIG. 8

Scanning electron micrograph of P. aeruginosa ATCC 27853 grown on glucose in the absence of rhamnolipid (A) and presence of 6 mM rhamnolipid (B) for 8 h. The scanning electron micrographs shown are of cells fixed with 1% rhenium tetroxide. Rhenium tetroxide was considered superior to osmium tetroxide because of its ability to preserve anything larger than a simple hexose structure. Thus, this fixative better preserves external cell morphology and prevents artifacts due to condensation of supernatant materials onto the cell surface.