Studies On The Effect Of Rhamnolipid Biosurfactant On Biodegradation Of Petroleum Hydrocarbon Contaminants

The hydrocarbon contamination of the environment is a serious threat to the health of human and ecosystem. Bioremediation has performed a great promise as a potentially effective and low-cost treatment option of hydrocarbon contamination. However, the low bioavailability of hydrocarbons leads to their slow biodegradation. An option to enhance the bioavailability of hydrocarbons is to add surfactants. Surfactants could increase the aqueous concentration of hydrophobic compounds resulting in higher mass transfer rate and bioavailability. But the use of synthetic surfactants in bio-treatment processes has been rather limited due to their toxicity and non-biodegradation. Biosurfactants have the advantages of being readily biodegradable and non-toxic compared to chemically produced surfactants which makes them more valuable in hydrocarbon biodegradation.In this study, biosurfactant and biosurfactant-producing microorganisms were investigated for their potential in enhancing bioavailiability and biodegradation of hydrocarbon. The main results were summarized as follows:Ⅰ. A strain which could highly produce biosurfactant was screened. The structure and physico-chemical properties of the biosurfactant produced by the strain were also studied intensively.1. A surfactant-producing bacterium, named as O-2-2, was screened from oil-producing wastewater and identified as Pseudomonas aeruginosa based on its morphological and physiological characteristics. The results of TLC, FT-IR and GC-MS showed that the biosurfactant produced by the strain O-2-2 was a rhamnolipid mixture.2. The optimal medium components and growth conditions for rhamnolipid production by the strain O-2-2 were studied using "one-variable-at-a-time" method. The results showed that the optimal medium for rhamnolipid production by this strain was 80.0 g/L peanut,10.0 g/L NaNO3, 2.0 g/L KH2PO4, 5.0 g/L K2HPO4, 0.2 g/L MgSO4-7H2O, 0.01 g/L CaCl2, 0.01 g/L FeSO4-7H2O, pH 7.0. The optimal cultivation conditions were observed at 32 °C and with 200 r/min. Under such conditions, the yield of rhamnolipid was as high as 19.34 g/L within 108 h.3. High pressure liquid chromatography/mass spectrometry using electrospray ionizationwas first used to analyze the components of rhamnolipid biosurfactant. 21 rhamnolipid homologs from the biosurfactant mixture produced by the strain 0-2-2 with peanut oil as carbon source were isolated and identified. The rhamnolipid homologs are formed by one or two rhamnose molecules linked to one or two /Miydroxy fatty acids of saturated or unsaturated alkyl chain between Cg and Cn-4. The critical micelle concentration of the biosurfactant is 70.5 mg/L. Its surface tension,interface tension (against n-hexadecane) and hydrophile-lipophile balance are 28.6 raN/m, 1.0 mN/m and 11.2, respectively. The biosurfactant can keep its surface activity at high temperature, in high-salinity solution and in a wide pH range of 5.5-14.0. Moreover, the biosurfactant possesses a high emulsifying activity to many kinds of hydrophobic organic substance, such as liquid paraffin, diesel oil and toluene.5. The solubilities of naphthalene, phenanthrene and pyrene increase linearly with rhamnolipid biosurfactant dose when the surfactant concentration is above CMC. The molar solubilization ratio (MSR) values decrease with increasing solute size and the micelle-phase/aqueous-phase partition coefficient (KmjC) values increase with the increasing hydrophobicity of PAHs. The phenanthrene solubility in rhamnolipid solution increases with the increase of temperature and salinity and reaches maximum at pH value of 5.5.II. The effect of rhamnolipid on biodegradation of alkane by P. earuginosa PS-1 was investigated. The biodegradation of n-hexadecane increased with obviously in the precence of the rhamnolipid. As viewed from the interactions between pollutant, hydrocarbon-degrading microorganisms and biosurfactant, the mechanisms of biosurfactant-enhanced biodegradation of hydrophobic hydrocarbon were intensivelyinvestigated. There are three mechanisms by which rhamnolipid biosurfactant enhanced the bioavailability of alkane. First, rhamnolipid can emulsify large oil droplets to small ones, therefore increasing substrate-water interfacial area and creating more opportunity for direct contract between cells and substrate. Second, rhamnolipid can promote alkane uptake through solubilization of hydrocarbon in micelles, which facilitates alkane into the cell through amphiphatic channel. Third, rhamnolipid can cause the cell surface to become more hydrophobic, which can increase the association of the cell with alkane substrate. In this study, the probable mechanism of the enhanced hydrophobicity of cell surface was concluded, which showed that biosurfactant can induce lipopolysacchalide to release from the cell surface by dissolution or complexation.III. The influence of rhamnolipid and sodium dodecyl sulfonate (SDS) on the phenanthrene biodegradation by Pseudomonas putida S-10-3 in aqueous and sediment slurry was studied. The artifically fresh-contaminated sediment and aged-contaminated one were used to determine the effect of aging process on bioremediation.1. Both rhamnolipid and SDS at high doses (above effective critical micelle concentration, CMCeff) can greatly enhance the desorption of sediment-sorbed phenanthrene, and rhamnolipid is more effective than SDS.2. The results of biodegradation experiments indicate that the biodegradation of phenanthrene depends on both the type of surfactants and their concentrations. During phenanthrene biodegradation, rhamnolipid was degraded by P. putida S-10-3 simultaneously, but SDS was not. Within optimal concentrations range of rhamnolipid, the biodegradation rate was enhanced with the increase of surfactant dose. There are two possible reasons, one is that rhamnolipid increase the solubility of phenanthrene and its bioavailability; the other is that the rhamnolipid may serve as cometabolic substance to increase biomass and promote the degradation rate of phenanthrene. However, with much higher dosage of rhamnolipid, the microorganisms might preferentially metabolize the biosurfactant and the phenanthrene biodegradation was inhibited. When amended with much higher of SDS, phenanthrene biodegradation was also inhibited because ofthe limited mass transfer of phenanthrene from micelle into water.3. With addition of surfactants into sediment slurry, the mass transfer of phenanthrene from sediment-sorbed phase to aqueous phase increased, resulting in the enhancement of phenanthrene biodegradation. On the other hand, aging process significantly decreased the desorption and biodegradation rate of pollutant.IV. The effects of adding biosurfactant and inoculating biosurfactant-producing strain to the petroleum hydrocarbons biodegradation were investigated.1. The addition of rhamnolipid increased the biodegradation extent of total petroleum hydrocarbon from 35.7% to 57.6% within 20 days. The biodegradation rates of both saturate and aromatic fractions were enhanced.2. Biosurfactant-producing strain 0-2-2 could utilize saturate fractions of crude oil and excrete rhamnolipid biosurfactant, resulting in a higher biodegradation of total petroleum hydrocarbon. Co-inoculating biosurfactant-producing strain could enhance the biodegradation of saturate fractions greatly, but decrease that of aromatic fractions, which indicated that there might be competition between biosurfactant-producing strain and the hydrocarbon-degrading consortia.