鼠李糖脂生物表面活性剂对石油烃污染物生物降解影响的研究
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摘要
环境中广泛存在的石油烃污染物对人体健康和生态系统的安全构成了很大威胁。生物修复因其经济有效而成为一种最有发展潜力的治理石油烃污染的技术。但石油烃污染物生物可利用性低是限制其生物降解的主要因素。添加表面活性剂是提高其生物可利用性的一个有效方法。表面活性剂能够增大疏水性有机物在水相中的溶解度从而增加传质速率和生物可利用性。但化学合成的表面活性剂本身有毒且难于降解,使其在石油污染生物治理中的应用受到限制。生物表面活性剂具有无毒且易于降解等特性,这使其在石油烃污染的生物治理中具有广泛的应用前景。
本论文考察了鼠李糖脂生物表面活性剂及其产生菌对石油烃污染物生物可利用性和生物降解的影响。主要研究结果可归纳如下:
1.筛选出生物表面活性剂高产菌并对该菌株所产表面活性剂的结构和理化性质进行了系统研究。
(1) 从含油废水中筛选出生物表面活性剂产生菌O-2-2,依据其生长形态及生理生化特征鉴定为铜绿假单胞菌(Pseudomonas aeruginosa)。利用薄层层析(TLC)、红外光谱(FT-IR)及气质联用(GC-MS)等手段分析表明该菌株所产生物表面活性剂为鼠李糖脂混合物。
(2) 采用单次单因子法,确定菌株O-2-2产鼠李糖脂的适宜培养基组成和适宜培养条件。结果表明该菌株产鼠李糖脂的适宜培养基组成为(g/L):花生油80.0,NaNO_310.0,NaCl 1.1,KH_2PO_4 2.0,K_2HPO_4 5.0,MgSO_4·7H_2O 0.2,CaCl_2 0.01,FeSO_4.7H_2O 0.01,pH 7.0。该菌株产鼠李糖脂的适宜培养条件为:温度32℃,摇床转速200r/min。在此条件下培养108h,鼠李糖脂的产率达19.34g/L。
(3) 利用高效液相色谱/电喷雾离子化质谱仪分离并鉴定了菌株O-2-2以花生油为碳源所产生物表面活性剂的组分。结果表明该生物表面活性剂中共含有21种鼠李糖脂的同系物,都由1～2分子的鼠李糖和1-2个含β-羟基的碳链长度为8～12的饱和或不饱和脂肪酸构成。
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 intensively
investigated. 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 of
the 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.
本论文考察了鼠李糖脂生物表面活性剂及其产生菌对石油烃污染物生物可利用性和生物降解的影响。主要研究结果可归纳如下:
1.筛选出生物表面活性剂高产菌并对该菌株所产表面活性剂的结构和理化性质进行了系统研究。
(1) 从含油废水中筛选出生物表面活性剂产生菌O-2-2,依据其生长形态及生理生化特征鉴定为铜绿假单胞菌(Pseudomonas aeruginosa)。利用薄层层析(TLC)、红外光谱(FT-IR)及气质联用(GC-MS)等手段分析表明该菌株所产生物表面活性剂为鼠李糖脂混合物。
(2) 采用单次单因子法,确定菌株O-2-2产鼠李糖脂的适宜培养基组成和适宜培养条件。结果表明该菌株产鼠李糖脂的适宜培养基组成为(g/L):花生油80.0,NaNO_310.0,NaCl 1.1,KH_2PO_4 2.0,K_2HPO_4 5.0,MgSO_4·7H_2O 0.2,CaCl_2 0.01,FeSO_4.7H_2O 0.01,pH 7.0。该菌株产鼠李糖脂的适宜培养条件为:温度32℃,摇床转速200r/min。在此条件下培养108h,鼠李糖脂的产率达19.34g/L。
(3) 利用高效液相色谱/电喷雾离子化质谱仪分离并鉴定了菌株O-2-2以花生油为碳源所产生物表面活性剂的组分。结果表明该生物表面活性剂中共含有21种鼠李糖脂的同系物,都由1～2分子的鼠李糖和1-2个含β-羟基的碳链长度为8～12的饱和或不饱和脂肪酸构成。
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 intensively
investigated. 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 of
the 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.
引文
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