【作者】 马满英；

【导师】 施周；

【作者基本信息】 湖南大学 ， 市政工程， 2009， 博士

【摘要】 在充分综述国内外相关文献的基础上,研究了生物表面活性剂鼠李糖脂的制备及其在修复多氯联苯（PCBs）污染土壤中的应用。通过单因素及正交实验对铜绿假单胞杆菌AB93066产鼠李糖脂（RL）的发酵培养基配方和产生规律进行了研究。结果表明最佳培养基配方为:ρ（酵母膏）=0.2g/L ,ρ（豆油）=120g/L ,ρ（NaNO₃）=6.5g/L ,ρ（KH₂PO4）=1.0g/L ,ρ（Na₂HPO₄·12_H2O）=1.0g/L,ρ（MgSO₄·7H₂O）=0.1g/L,ρ（FeSO₄·7H₂O）=0.2g/L,pH=6.0。RL的最佳收获期在发酵后156h～168h。发酵生产RL的扩大试验表明,在最优发酵条件下RL的产量可达56g/L以上,提取后的RL可将去离子水的表面张力降至29.01mN/m。用高效液相色谱/质谱联用仪进一步分析了所提取的RL的组成,其分别为二鼠李糖脂（R1）和单鼠李糖脂（R2）两种同系物。R1和R2的临界胶束浓度（CMC）分别为0.03mmol/L和0.04mmol/L。通过正交优化实验找到了一种新的RL提取方法,即预处理酸沉淀冷冻干燥法。与传统方法提取的RL相比,新方法提取的RL纯度更高,且其产量提高了8.8g/L,CMC降低了5mg/L;传统工艺需要大量的氯仿和甲醇等有毒溶剂,而新工艺不需要这些有毒物质。RL对PCBs的增溶作用主要是由于表面活性剂胶束作用的结果,随PCBs分子中氯含量的增大而减小。RL对PCBs的增溶作用要高于三种非离子化学表面活性剂POE（6）、POE（10）和Brij35。PCBs在RL溶液中的胶束相/水相分配系数（Kmc）随PCBs的疏水性增强而增大。PCBs的辛醇/水分配系数的对数logKOW与logKmc之间的线性回归方程为:logKmc=0.48logKOW+3.08。PCBs在共溶时的表观溶解度小于其作为单一溶质时的表观溶解度。适当增加Na+、Mg²⁺、Ca²⁺的浓度对RL溶液增溶2,2’,4,4’CB有显著的促进作用,但Ca²⁺不宜超过0.2mmol/L。无机阴离子Cl-、NO3-、SO42-对RL溶液增溶2,2’,4,4’CB的影响很小。RL与非离子表面活性剂对PCBs的增溶存在协同效应,其协同增溶作用的大小与其中非离子表面活性剂的HLB值呈正相关。pH对RL增溶2,2’,4,4’CB的影响与RL的浓度有关,当ρ（RL）=300mg/L时,随着pH从5.5上升到8.0,RL对2,2’,4,4’CB的增溶作用逐渐减弱;当ρ（RL）=2000mg/L,溶液的pH从5.5升高到7.0时,2,2’,4,4’CB的溶解度变化很小,但当pH从7.0升高到9.0时,2,2’,4,4’CB的溶解度逐渐提高。当RL的浓度大于CMC时,RL对PCBs的洗脱有显著的促进作用。具有较低HLB的R2对PCBs的洗脱效果要优于R1。人工污染土壤中PCBs的洗脱效果要高于陈化土壤。污染土壤中TOC的含量越高,PCBs的洗脱率越低。延长解吸时间和增加洗脱次数可增加土壤中PCBs的洗脱率,但解吸时间采用48h较为合适,洗脱总次数以3次为佳。碱性环境（pH>7）或适当增加RL溶液中Na⁺、K⁺、Mg²⁺、Ca²⁺的浓度均有利于土壤中PCBs的洗脱,但Mg²⁺不宜大于0.8mmol/L,Ca²⁺不宜大于0.25mmol/L。土壤对RL的吸附实验数据与Langmuir等温吸附线有较好的拟合。在柱洗脱试验中,RL对人工污染土样的洗脱率大于60%,而对陈化土样的洗脱率不到20%。RL与POE（6）复配对人工污染土壤中PCBs的洗脱具有明显的协同作用。表面活性剂对PCBs的增溶作用是人工污染土壤中PCBs洗脱的主要机理;而表面活性剂对PCBs的增流作用是陈化土壤中PCBs洗脱的主要机理。增加复配试剂RL-POE（6）的浓度,可提高陈化土壤中PCBs的洗脱率。PCBs降解菌P.LB400在以RL、POE（6）和联苯为碳源的三种驯化培养基中均能够快速生长,但P.LB400利用联苯的能力最强;利用RL的能力次之。P.LB400菌在生长细胞降解体系中对PCBs的总生物降解率要高于休眠细胞降解体系。以联苯为碳源时,PCBs的生物降解率最高;以RL为碳源时,PCBs的生物降解率次之,但在生长体系中,与以联苯为碳源时的生物降解率非常接近。在休眠细胞降解体系中,P.LB400细胞不能充分利用RL作为碳源,RL对PCBs的生物降解有一定的抑制作用;而在生长细胞降解体系中,P.LB400细胞能够充分利用RL作为碳源,降解菌的生物量明显增长,RL对PCBs的生物降解具有显著的促进作用。在含有混合表面活性剂RL-POE（6）的土壤洗脱液中,PCBs的总生物降解率比仅含RL的土壤洗脱液中PCBs的总降解率略低。紫外光预照射对土壤洗脱液中剩余PCBs的生物降解有一定的促进作用。光照射和生物降解的耦合有利于提高PCBs的降解速率,光降解产物并不抑制微生物对剩余PCBs的利用。更多还原

【Abstract】 On the basis of summarizing the related references home and abroad, a great deal of experiments had been done to study the production of rhamnolipid biosurfactant and the application of rhamnolipids in the remediation of polychlorinated biphenyls（PCBs）-contaminated soil.The optimized conditions for fermentation medium and the production regulation of rhamnolipids（RL） produced by pseudomonas aeruginosa AB93066 were as follows. The composition of the medium wasρ（yeast）=0.2g/L,ρ（bean oil）=120g/L,ρ（NaNO₃）=6.5g/L,ρ（KH₂PO₄）= 1.0 g/L,ρ（Na₂HPO₄·12H₂O）=1.0g/L,ρ（MgSO₄·7H₂O）=0.1g/L andρ（FeSO₄·7 H₂O）=0.2g/L, pH=6.0. The time range for harvesting RL was between 156h and 168h after fermentation. Scale-up production experiment indicated that RL yield was 56g/L and more under the optimal conditions. Surface tension measurements showed that the extracted RL could decrease the surface tension of deionized water to 29.01mN/m. Further, dirhamnolipid（R1） and monorhamnolipid（R²） were separated and identified from the extracted RL using liquid chromatography/mass spectrometry. The critical micelle concentration （CMC） of R1 and R2 were 0.03mmol/L and 0.04mmol/L, respectively. A new procedure of pretreatment/acid precipitation/ freeze -dryness for rhamnolipid extraction was determined by orthogonally designed experiments. That RL extracted through the new procedures was purer, the yield was 8.8g/L higher and the CMC was 5mg/L lower than that extracted through the conventional procedures. Large amount of harmful solvents such as chloroform and methanol were needed in the conventional procedures and no such toxic solvents were used in the new procedures for the RL extraction.Those surfactant micelles played a main role in solubilization of PCBs by RL. The apparent aqueous solubility of PCBs in RL solution decreased with increasing chlorination of the PCB molecule. RL was more effective for solubilization of PCBs than three nonionic chemical surfactants, such as POE（6）, POE（10） and Brij35. Micelle-phase/aqueous -phase partitioning coefficient (K_mc) of PCBs in RL solution increased with decreasing intrinsic solubility of PCBs in pure water.The relationship between Octanol/water partitioning coefficient（KOW） and Kmc was described by linear repression analysis as following: logKmc=0.48logKOW+3.08. The apparent aqueous solubility of individual congener in PCB mixture in RL solution was much lower than the solubility when individual congener was the sole contaminant in RL solution. Na+、Mg²⁺、Ca²⁺ within optimal concentrations range could obviously enhance the apparent aqueous solubility of 2,2’,4,4’CB in RL solution, but Ca²⁺ greater than 0.2mM was ineffective. Inorganic anion（ such as Cl-, NO3-, SO42-）had little affection on the solubilization of 2,2’,4,4’CB by RL. PCBs were synergistically solubilized by RL and nonionic surfactant. The synergistic power of the mixed surfactants for PCBs was positively relative to hydrophile-lipophile balance（HLB） of nonionic surfactant. Impact of pH on the solubilization of 2,2’,4,4’CB by RL was relative to the concentration of RL. At a RL concentration of 300mg/L, the aqueous solubility of 2,2’,4,4’CB in RL solution decreased when the pH changed from 5.5 to 8.0. At a RL concentration of 2 000mg/L, the aqueous solubility of 2,2’,4,4’CB had little change when the pH changed from 5.5 to 7.0, but the aqueous solubility increased with pH increasing from 7.0 to 9.0.Rhamnolipids obviously enhanced PCBs’desorption from soils when their concentrations exceeded CMC. Rhamnolipid R2 with lower HLB was more effective for PCBs’desorption than rhamnolipid R1. The desorption percentage of PCBs from laboratory contaminated soil was higher than that from field contaminated soil. The greater the TOC of soils, the lower the desorption precentage of PCBs. The desorption percentage of PCBs increased as the increase of desorption duration, wash times, pH （above 7）, and 48h of desorption duration, and 3 repeat washings were the most effective for the soil washing under the test conditions. Inorganic cationic, such as Na⁺, K⁺, Mg²⁺, Ca²⁺ within optimal concentrations range could obviously enhance desorption of PCBs from soil, and Ca²⁺ greater than 0.25mM or Mg²⁺ greater than 0.8mM was ineffective for increasing desorption of PCBs from soil. The Langmuir isotherm appeared to better describe binding of RL to the soils in the study. The column-washing experiment results showed: 60% of the sorbed PCBs were removed from laboratory contaminated soil using RL solution, but only 20% of the sorbed PCBs were removed from field contaminated soil. PCBs from laboratory contaminated soil were synergistically desorbed by RL and nonionic surfactant. The desorption of PCBs from laboratory contaminated soil was mainly attributed to solubilization of PCBs by RL, but the desorption of PCBs from field contaminated soil was mainly attributed to mobilization of PCBs by RL. Increasing the concentration of binary mixed-surfactants （RL-POE（6）） leaded to increasing precentage of PCBs washed from field contaminated soil.Pseudomonas strain LB400, a PCB specific microorganism, was able to grow rapidly in three culture media with RL, POE（6） or biphenyl as carbon source. The culture media with biphenyl present in solution yielded the highest cell density, while the cell density in culture media containing RL was ranked the second. Biodehradation rate of PCBs in growing cell system was greater than that in resting cell system. Biodegradation rate of PCBs with RL as carbon source was less than that with biphenyl as carbon source, but in growing cell it was very close to that with biphenyl as carbon source. In resting cell system, P.LB400 was not able to fully utilizing RL as carbon source, so RL had negative impact on Biodegradation of PCBs. In growing cell system, P.LB400 was able to fully utilizing RL as carbon source and the cell density of P.LB400 rapidly increased with increasing the concentration of RL, so RL obviously promoted Biodegradation of PCBs. Biodegradation rate of PCBs in the soil wash fluid containing surfactants mixed by RL and POE（6） was a little less than that in the soil wash fluid only containing RL. Precedent photolysis was able to improve Biodegradation of PCBs that remained in the soil wash fluid. A combination of UV pre-irradiation and biological treatment was beneficial to increasing degradation rate of PCBs. The products of photolysis did not inhibit biodegradation of the remaining PCBs.更多还原