The Mechanism Study For Rhamnolipid-mediated Biodegradation Of Petroleum Hydrocarbons Contaminants

The rapid development of energy exploitation and industrial caused serious petroleum hydrocarbons pollution,which brings huge damages to human health and the ecological environment and has aroused word wide attention.Bioremediation technology has the very high ecological significance for the environmental remediation.However,the strong hydrophobic characteristic of hydrocarbon makes them usually exist as the form of non-aqueous phase liquids,resulted in the low bioremediation efficiency.The biosurfactant plays an important role in the biological remediation;however,the underlying mechanisms have not been well studied.In this paper,the effects of biosurfactant on the biodegradation of petroleum hydrocarbons were investigated using rhamnolipid as the model biosurfactant.The effect of rhamnolipids on the surface hydrophobicity of the bacterial cells was systemly investigated.This biological effect of rhamnolipid on the biodegradation of petroleum hydrocarbons was also studied.Based on the above works,the biodegradation of n-hexadecane in a variety of forms was investigated,and the relationship between the solubilization and the bioavailability of petroleum hydrocarbons was further investigated.These works provide theoretical guidance for the effic ient application of biosurfactant in remediation of petroleum hydrocarbon pollution.The specific research work and achievements of this paper include the following five parts:The first part introduces the purification methods of rhamnolipid and the formation of rhamnolipid/alkane aggregates.Column chromatography and thin-layer chromatography were used to purify the crude products of rhamnolipid,and two separate groups of monorhamnolipid and dirhamnolipid were obtained.HPLC-MS examination showed that monorhamnolipids contained three major components:RhaC₁₀C₈,RhaC₁₀C₁₀,and RhaC₁₀C₁₂,while RhaC₁₀C₁₀0 also existed in dirhamnolipid.Results of the study suggest that column chromatography technique is effective in depth purification of rhamnolipid.The data of hexadecane aqueous dispersion showed that rhamnolipid could solubilize hexadecane when the concentration was below critical micelle concentration?CMC?,and the observation of cryo-TEM found that there is the formation of aggregates in this solution.In the second part,degradation of rhamnolipid-solubilized hexadecane and mass hexadecane as a separate phase by P.aeruginosa CCTCC AB93066 treated with rhamnolipid biosurfactant was studied for better understanding on the biological effect of rhamnolipid on hydrocarbon biodegradation.Different concentrations of Rhamnolipid were used for treating cells in degradation experiment.Results showed that 1CMC rhamnolipid treatment had a small inhibitory effect on cell growth.However,10 CMC rhamnolipid treatment accelerated degradation of mass hexadecane.It is possible that the treatment of rhamnolipid enhance the interfacial uptake of hexadecane or the permeability of cell surface.No degradation of 10 CMC rhamnolipid-solubilized hexadecane was observed for the cells treated with or without rhamnolipid,indicating that the pseudo-solubilized hexadecane is not available to the bacteria cells.These results are importance for evaluation of rhamnolipid biosurfactant application to hydrocarbon-contaminated sites during remediation.In the third part,degradation of hexadecane by P.aeruginosa ATCC 9027mediated with rhamnolipid?octylphenol ethoxylate?Triton X-100?or sodium dodecylbenzene sulfonate?SDBS?was studied to examine the effect of surfactant pseudo-solubilization on hexadecane biodegradation.The solubilizing ability of surfactants are followed Triton X-100>rhamnolipid>SDBS.The presence of rhamnolipid significantly increased the biodegradation efficient of hexadecane,which is because rhamnolipid solubilization increased the bioavailability of hexadecane for this bacterium.Although the biodegradation of Triton X-100 enhanced the growth of bacteria,it delayed the uptake of hexadecane by cells.For SDBS,the degradation of hexadecane in pseudo-solubilized form was decreased compared to hexadecane as a separate phase,showing an inhibitory effect on degradation of pseudo-solubilized hexadecane.It is inferred from the data that the effectiveness of surfactant to accelerate degradation of hydrocarbons by enhancing solubilization of the hydrocarbons may not always be guaranteed.In the fourth part,liquid culture systems containing rhamnolipid-solubilized,separate-phase,and multi-state hexadecane as the carbon source were employed for examining the effect of rhamnolipid solubilization on the bioavailability of hexadecane.Experimental results showed that the uptake of rhamnolipid-solubilized hexadecane by P.aeruginosa ATCC 9027,a rhamnolipid producing strain,was enhanced compared to the uptake of mass hexadecane as a separate phase,indicating rhamnolipid solubilization increased the bioavailability of hexadecane for this bacterium.For P.putida CICC 20575 which does not produce but degrade rhamnolipid,the uptake of either rhamnolipid-solubilized hexadecane or multi-state hexadecane was inhibited.The results show that how rhamnolipid solubilization changes the bioavailability of hexadecane depends on the bacterial compatibility to rhamnolipid.The study adds insight into the knowledge of biosurfactant-associated bioavailability of hydrophobic organic compounds?HOCs?,and is of importance for application of biosurfactants in bioremediation of HOCs.