Limited Role of Rhamnolipids on Cadmium Resistance for an Endogenous-Secretion Bacterium

Abstract

Rhamnolipids, a type of biosurfactant, represent a potential strategy for both enhancing organismic resistance and in situ remediation of heavy metals contaminations. In-depth study of the mechanism of rhamnolipids synthesis in response to heavy metals stress, is indispensable for a wide use of biosurfactant-secreting microbes in bioremediation. In this study, we employed the wild-type and the rhlAB deficient strain (ΔrhlAB) of Pseudomonas aeruginosa, a prototypal rhamnolipids-producing soil microorganism, to investigate its responses to cadmium resistance based on its physicochemical, and physiological properties. Compared with the wild-type strain, the ΔrhlAB were more sensitive to Cd-stress at low Cd concentration (<50 mg/L), whereas there was little difference in sensitivity at higher Cd concentrations, as shown by spot titers and cell viability assays. Secreted rhamnolipids reduced intracellular Cd²⁺ accumulation to alleviate Cd²⁺ stress, whereas endogenous rhamnolipids played a limited role in alleviating Cd²⁺ stress. Synthesized rhamnolipids exhibited a higher critical micelle concentration (CMC) (674.1 mg/L) and lower emulsification index (4.7%) under high Cd-stress, while these parameters showed no obvious changes. High Cd-stress resulted in high hydrophilic wild-type bacterial surface and lower bioremediation ability. This study could advance a deeper understanding of the mechanism of cadmium resistance and provide a theoretical foundation for the application of biosurfactant and biosurfactant-secreted bacterium in contaminant bioremediation.

Keywords: biosurfactants characteristics; cadmium stress; cytotoxicity reduction; endogenous rhamnolipids.

Figure 1

Construction of the ΔrhlAB strain using the two-step allelic exchange by pEX18GM (A), cell viability tests (B), and spot titer assays (C) of P. aeruginosa wild-type and ΔrhlAB strains under a series of Cd concentrations in the LB media. Differences were considered significant at p < 0.05, and * presented p < 0.05, ** presented p < 0.01, *** presented p < 0.005.

Figure 2

Intracellular cadmium amount per dry biomass (A), bacterial hydrophobicity (B), and SEM images (C) for wild-type and ΔrhlAB strains exposed to a series of Cd concentrations. Differences were considered significant at p < 0.05, and * presented p < 0.05, *** presented p < 0.005.

Figure 3

Bacterial membranes damaged (MDA, A), reactive oxygen species (ROS, B), reductive glutathione (GSH, C), and total sulfhydryl group (Total-SH, D) amounts for wild-type and ΔrhlAB strains. Differences were considered significant at p < 0.05, *** presented p < 0.005.

Figure 4

Rhamnolipids secretion (A), surface tensions (B), and emulsification ability tests (C) for the wild-type strain with different Cd concentrations. Differences were considered significant at p < 0.05, and * presented p < 0.05, ** presented p < 0.01.

Figure 5

Critical micelle concentrations (A, B, and C for 0, 5, and 200 mg/L Cd²⁺, respectively) of rhamnolipids extracted from wild-type cells with different Cd concentrations; schematic diagram for the effect of Cd²⁺ on wild-type bacterial cells and rhamnolipids; words in red represent enhancement, blue words represent reduction (D).