Adsorption Of Rhamnolipid Biosurfactant On Microorganisms And The Effect Of The Adsorption On Cell Surface Hydrophobicity

In soil remediation, one of the effects of biosurfactants to enhance degradation of hydrophobic organic contaminants is to increase the microbial cell surface hydrophobicity and facilitate the direct contact of microorganisms with the contaminants trapped in soil pores. However, the manner of interaction between biosurfactants and microorganisms as well as the rules of cell surface hydrophobicity change as the function of biosurfactants were not fully understood. In this paper, the adsorption of the rhamnolipid biosurfactant on microorganisms and change of cell surface hydrophobicity due to the adsorption was systematically studied. Effect of the adsorption on microbial degradation of different carbon sources was also investigated.First the rhamnolipid biosurfactant was produced by aerobic fermentation using a Pseudomonas aeruginosa strain. It was separated from the culture by acid precipitation and purified by column chromatography until monorhamnolipid and dirhamnolipid were obtained. HPLC-MS examination showed that both of the rhamnolipids contained three main components and they were used in the following studies.Next aggregation behavior of the monorhamnolipid and dirhamnolipid in mineral salt medium (MSM) around neutral pH was investigated as the support for studies on microbial adsorption of them. The results showed that their critical micellar concentrations in MSM at pH 6.5 were 75μM and 106μM, respectively. Both of the rhamnolipids could form multi-size aggregates in the solution, but they were different in size and distribution. For both of the rhamnolipids, the size of the aggregates decreased with increasing of surfactant aqueous concentration when it was lower than CMC, and within the whole concentration range volume distribution of big aggregate forms decreased and that of the small form increased. Increasing pH of the solutions caused decrease of the aggregate size for both rhamnolipids and transformation of big to small. The size of the aggregates was assumed to be a function of tightness of rhamnolipid molecular layer and the surface charge of the aggregates, which was consolidated by change of surface zeta potential of dirhamnolipid aggregates with surfactant concentration and solution pH.Next adsorption of dirhamnolipid and other four synthetic surfactants on Pseudomonas aeruginosa cells, adsorption of dirhamnolipid on the cells of four microorganisms in different physiological status, and adsorption of monorhamnolipid and dirhamnolipid on cells of two Pseudomonas aeruginosa strains were consecutively studied, and in each experiment the adsorption-induced change of cell surface hydrophobicity was investigated. The objective was to find the adsorption rules and reveal the adsorption mechanism considering both adsorbate and adsorbent, as well as to disclose the relation between the adsorption and the change of cell surface hydrophobicity. Result of the first experiment showed that the adsorption kinetics of all the surfactants on the Pseudomonas aeruginosa cells followed the second-order law, and the adsorption isotherms of all the surfactants on the bacterium fitted Freundlich equation well. The strength of the adsorption was highly related to the surfactant molecular structure and size. The adsorption mode for the surfactants was probably hydrophilic interaction such as electrostatic attraction, because except for SDS the adsorption totally turned the cell surface to be more hydrophobic. Result of the second experiment showed that the adsorption was not only specific to the microorganisms but also depended on the physiological status of the cells. e.g. adsorption of the dirhamnolipid on the exponential-phase cells of Pseudomonas aeruginosa CCTCC AB93066, Bacillus subtilis CCTCC AB93108, and Candida lipolytica CCTCC AB92044 was stronger than on their stationary-phase cells, but reversed result was found for Bacillus subtilis CCTCC N1. The secondary adsorption, which was supposed to be adsorption of rhamnolipid aggregates, was found for most of the cells. Components of the biosurfactant with different aliphatic chain length also exhibited slight difference in adsorption manner. The adsorption totally caused the cell surface hydrophobicity to change regularly; however, the changes depended on the type and physiological status of the microorganism, as well as the concentrations of rhamnolipid solutions applied. Orientation of rhamnolipid monomers on cell surface was supposed to be the basic means of adsorption to change cell hydrophobicity, which was more remarkable at low rhamnolipid concentrations. Result of the third experiment showed that the adsorption capacity of all the cells to monorhamnolipid was much stronger than to dirhamnolipid, indicating that the structure of polar moiety of rhamnolipid has strong effect on the adsorption. Although both bacteria were Pseudomonas aeruginosa, the difference between strains also caused different adsorption manner in that the rhamnolipid-sourced P. aeruginosa cells had weaker adsorption capacity to dirhamnolipid or even released extra dirhamnolipid when aqueous concentration of the surfactant is high enough. The difference in the carbon source to grow the bacteria had little influence on the adsorption. Adsorption of both monorhamnolipid and dirhamnlipid tended to turn the cell surface more hydrophobic and the effect of dirhamnolipid was stronger.Finally the ability of the two rhamonolipid to enhance the apparent solubility of n-hexadecane was investigated, and the effect of monorhamnolipid adsorption on degradation of glucose, aggregate-incorporated n-hexadecane and single-phased n-hexadecane was also studied. The monorhamnolipid exhibited different manner of enhancing hexadecane solubility when its aqueous concentration was lower or higher than CMC, while the capability of dirhamnolipid was the same in the whole concentration range tested. Adsorption of monorhamnolipid of low concentration to some extent restricted the cell growth on glucose and single-pahsed hexadecane, however, adsorption of monorhamnolipid of high concentration had insignificant or stimulative effect on the cell growth on glucose and on hexadecane, respectively. For the hexadecane incorporated in aggregates formed by high-concentration monorhamnolipid, they could not be degraded by the cells treated with or without the surfactant. This result indicated that the incorporated hexadecane was unavailable to the Pseudomonas aeruginosa strain, though scanning electron microscope showed that the aggregates adsorbed to the cells.The paper disclosed the rules of adsorption of rhamnolipid biosurfactants to microorganisms and demonstrated the effect of adsorption to change cell surface hydrophobicity. Because this effect occurs at low biosurfactant concentrations, it implies the possibility of economical and effective application of biosurfactant in soil remediation, especially in situ remediation with exogenous bacterial amendments. In order to make the cell surface hydrophobicity controllable using biosurfactant, the mechanism of interaction between biosurfactant molecules and cell surface chemical groups should be further studied. On the other hand, the interactions between the biosurfactant-modified cells, soil colloids and contaminants should be investigated for successful application of the technology.