生物表面活性剂复配行为及在疏水性有机污染修复中的应用
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摘要
表面活性剂增效修复是一种很有前途的去除土壤及地下水中疏水性有机污染物技术。相比于化学合成表面活性剂，生物表面活性剂具有高表（界）面活性、低临界胶束浓度（CMC）、低毒、易于生物降解等特点，在疏水性有机物污染场地修复中具有良好的应用潜力。在实际应用中，往往将不同类型表面活性剂复配，利用混合表面活性剂较单一表面活性剂所具有的更强的增溶能力和更好的胶体稳定性，来提高修复效率，降低修复成本。但目前缺乏对生物表面活性剂特别是其复配体系所形成胶束结构和胶束基本性质的认识，限制了应用生物表面活性剂及其复配体系增溶洗脱疏水性有机物的深入研究，因而难以指导生物表面活性剂及其复配体系在疏水性有机污染场地的应用。
本论文在选育槐糖脂生物表面活性剂高产菌和确定槐糖脂主要理化性质的基础上，综合应用稳态荧光探针法、表面张力法、动态激光散射法和低温透射电镜等方法，系统研究了非离子生物表面活性剂槐糖脂与阴离子生物表面活性剂鼠李糖脂复配体系的胶束形态、胶束结构及热力学和动力学性质，进而探讨了该复配体系对多环芳烃（PAHs）污染土壤增效修复作用及其机制，并应用均匀设计法，开发出以糖脂类生物表面活性剂和非离子的山梨醇酯类表面活性剂复配体系为主体的环境友好型溢油分散剂。论文主要研究结果可归纳如下：
1.筛选出槐糖脂生物表面活性剂高产菌，确定了该菌株利用不同碳源所产槐糖脂同系物的组成和主要理化性质，优化了该菌株利用不同底物发酵制备槐糖脂的培养基组成。
（1）从含油废水中筛选出生物表面活性剂高产菌O-13-1，经18S rDNA测序鉴定为假丝酵母菌（Starmerella bombicola），所产生物表面活性剂经薄层层析（TLC）、红外光谱（FT-IR）及液质联用（HPLC-MS/MS）确定为槐糖脂同系物。该菌株以葡萄糖为水溶性底物和以油酸、菜籽油、棉籽油和煎炸废油等脂溶性底物为双碳源发酵时，产物均为含多种自由酸型和内酯型槐糖脂同系物，而内酯型同系物为主要成分，在槐糖内酯中均以脂肪烃链为单不饱和的十八烯酸（C18:1）结构为主，如ω-1型17-L-[(2'-O-β-D-吡喃葡萄糖基-β-D-吡喃葡萄糖基)-O-]-十八烯酸-1',4"-内酯-6',6"二乙酸酯。
（2）槐糖脂溶液具有良好的表面活性、较低的临界胶束浓度（CMC）和良好的耐温性、耐酸碱性和热稳定性。该糖脂溶液能使水的表面张力将至37.0mN/m，CMC仅为30mg/L，在100℃下加热2h、NaCl浓度为0-20%和pH为2-10范围时均保持较高表面活性；而槐糖内脂的CMC值随短链脂肪醇的加入而增大，随NaCl电解质的加入而减小。
（3）通过设计正交试验，优化确定O-13-1菌株产槐糖脂的摇瓶发酵培养基组成为：葡萄糖60g/L；油酸60mL/L，酵母粉8g/L，尿素2g/L。而该菌株利用廉价水溶性碳源如葡萄糖浆和糖蜜以及脂溶性碳源如棉籽油和煎炸废油时槐糖脂产量较低；而以葡萄糖和油酸或葡萄糖和菜籽油作为双碳源时，发酵制备槐糖脂的碳源成本较低，分别为11040元/吨和11425元/吨。因此，O-13-1菌株发酵合成槐糖脂的优选水溶性碳源为葡萄糖，优选脂溶性碳源为油酸和菜籽油。
2.综合应用多种技术手段研究了槐糖脂和鼠李糖脂复配体系混合胶束和表面吸附性质。
（1）首先，采用稳态荧光探针方法测定了非离子生物表面活性剂槐糖内酯（LS）单组分和阴离子生物表面活性剂双鼠李糖脂（R2）单组分按比例复配所形成的混合胶束的CMC，并通过Rubingh规则溶液理论模型计算了二者相互作用参数β及热力学函数。LS/R2混合体系的CMC低于根据Clint理论混合模型计算的CMCideal值，在LS摩尔分数（αLS）=0.1时，该复配体系的协同增效作用最为明显，混合体系的CMC值较单一LS或R2体系的CMC值分别降低了61.7%和18.9%。αLS在0.025-0.3之间时，复配体系胶束相互作用参数β以及混合胶束形成自由能ΔGmic和超额自由能GE值更负，说明在溶液中有少量LS存在时，LS和R2在胶束中有强烈的吸引作用，易于自发形成混合胶束。
（2）鉴于实际发酵得到的槐糖脂和鼠李糖脂都为含有多种组分的同系物，本文进一步采用表面张力方法研究了槐糖脂同系物（SLs）和鼠李糖脂同系物（RLs）复配体系特点，通过吉布斯公式和Rubingh规则溶液理论模型计算了二者在表面吸附层和胶束中行为。结果显示复配体系的CMC实验值低于CMCideal，当溶液中含有少量的槐糖脂时，β更负，SLs和RLs在混合胶束中表现出强烈的吸引作用，与二者单体复配体系研究结果一致。槐糖脂在表面吸附层中的摩尔分数（xSLsσ）及混合胶束中的摩尔分数（xSLs）随其在溶液中摩尔分数（αSLs）的增大而增大且均大于αSLs，槐糖脂在表面吸附层和混合胶束中占优，对复配增效作用贡献较大。
（3）稳态荧光探针方法、动态激光散射法和低温透射电镜测定结果表明，在两种糖脂总浓度为0.3-0.8mmol/L范围时，SL/R2复配体系单分散胶束的Nagg仅为3-6，平均胶束直径约5-20nm，SL/R2复配体系所形成胶束具有聚集数（Nagg）小而胶束直径大的特点。同时在糖脂体系中，单分散胶束发生聚集形成胶束聚集体，当LS摩尔分数为0.7时，胶束聚集体直径最大，达160nm。这是由于槐糖内脂和双鼠李糖脂分子中均含有葡萄糖基和长链疏水基，占据较大空间，不利于分子间的有序排列，导致其复配体系形成尺寸较大的囊泡状胶束聚集体结构，可能对疏水性有机物具有较强增溶能力。
3.研究了SLs/RLs复配体系对水溶液中PAHs的增溶作用及其对PAHs污染土壤的淋洗修复作用，并考察了pH值、盐度和重金属离子等对SLs/RLs复配体系增效修复PAHs污染土壤的影响。
（1）SLs/RLs复配体系对水溶液中PAHs的增溶作用和对土壤中PAHs的洗脱作用明显优于二者单一存在的体系，且对于疏水性相对较弱的菲的增溶和解吸能力大于疏水性较强的芘。如在SLs摩尔分数为0.7时，SLs/RLs复配体系对PAHs的协同增溶作用最强，对菲的摩尔增溶比（MSR）为0.274，分别是SLs和RLs单一体系的1.6倍和8.6倍，对芘的MSR为0.079，分别为SLs和RLs单一体系的1.8和5.3倍。2种糖脂总浓度为4.2mmol/L且SLs摩尔分数为0.7时，SLs/RLs复配体系协同去除菲和芘的效率最高，分别达到71.5%和59.3%，较单一的SLs体系分别高出39.7%和35.7%，而较单一的RLs体系分别高出33.2%和32.7%。
（2）溶液pH值对SLs/RLs复配体系增溶PAHs能力有显著影响，在pH为5.5-8.0的范围内，其增溶能力随pH值的增大而减小，在pH为5.5时，SLs/RLs形成的混合胶束对PAHs的增溶效率最高。盐度对SLs/RLs复配体系洗脱PAHs作用的影响与表面活性剂浓度大小和盐度大小密切相关。两种糖脂总浓度小于3mmol/L时，洗脱液中添加0.01mol/L NaCl对SLs/RLs复配体系洗脱PAHs具有抑制作用，而两种糖脂总浓度大于4mmol/L时，洗脱液中添加0.01mol/L NaCl则对SLs/RLs复配体系洗脱PAHs具有促进作用；当洗脱液中添加0.05mol/LNaCl时，对SLs/RLs复配体系洗脱PAHs都具有抑制作用，但抑制作用明显小于单一的槐糖脂和鼠李糖脂体系，表明SLs/RLs复配体系具有更强的耐盐性；而共存重金属Cd2+对于槐糖脂、鼠李糖脂及复配体系解吸PAHs能力的影响较小。
4.将槐糖脂和鼠李糖脂复配体系应用到溢油分散剂制备中，开发出高效低毒的环境友好型溢油分散剂，并确定了该溢油分散剂的适宜应用条件。
（1）应用均匀实验设计方法，以分散率为主要评价指标，确定了以槐糖脂、鼠李糖脂、以及两种山梨醇酯类非离子表面活性剂复配体系为主体的浓缩型溢油分散剂配方SRTG-16。该配方为槐糖脂/鼠李糖脂/山梨醇酯1/山梨醇酯2/溶剂：6.80/0.83/3.28/39.10/50，该溢油分散剂对QHD32-6原油的分散性能优于任一单一表面活性剂，10min分散率达到46.30%。
（2）确定了该溢油分散剂的适宜应用条件为：剂油比，1:10-1:25；温度，5-20℃；盐度，0-40；pH，6.0-10.0。
（3）分别以斑马鱼（Danio rerio）和纹缟鰕虎鱼（Tridentiger trigonocephalus）为受试生物，通过急性毒性试验确定所开发的溢油分散剂SRTG-16的生物毒性，结果表明该溢油分散剂对斑马鱼（D. rerio）的48h致死率和对纹缟鰕虎鱼（T.trigonocephalus）的24h致死率都为0%，明显优于国标（GB18181.1-2000）中半致死时间大于24h的指标，表明该溢油分散剂具有较低的生物毒性。
（4）该溢油分散剂的BOD/COD值为33.8%，满足国标（GB18181.1-2000）中大于30%的要求，表明该溢油分散剂具有良好的生物可降解性。
上述结果表明所开发的溢油分散剂适用于海面溢油污染修复和治理。
Surfactant enhanced remediation (SEM) has been developed to be a promisingremediation method for the removal of hydrophobic organic compounds (HOCs) fromgroundwater and soil. Recently, the use of a wide range of surfactants in SEMremediation technology are synthetic surfactants. Biosurfactants have gained growinginterest due to their distinct advantages over their synthetic counterparts, such asspecificity, biodegradability, nontoxicity, and a broad range of structures. Mixedsurfactant systems often present superior properties to those with a single surfactantcomponent, such as better efficiency in decreasing surface/interfacial tensions, lowercritical micelle concentration (CMC), higher solubilizing power, and expandedcolloidal stability. Therefore, mixed surfactant systems, especially biosurfactantmixed systems, have good prospects of application for the remediation ofhydrophobic organics contaminated site. However, few recent studies havehighlighted the micelle structure and properties of biosurfactants, especially mixedbiosurfactants. The limited number of works involving these aspects has prevented adeeper knowledge and understanding of biosurfactants application insurfactant-enhanced remediation of hydrophobic organics contaminated. Therefore, itis difficult to guide the application of biosurfactants and mixed bisurfactant forremediation of hydrophobic organics contaminated site.
In this study, sophorolipid production strain with high yields was first screenedand main physicochemical properties were determined. Micellar and surfaceproperties of sophorolipid and rhamnolipid mixed systems were studied bysteady-state fluorescence spectroscopy, surface tension measurements, dynamic lightscattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM). Then,sophorolipids and rhamnolipids mixed systems were investigated for their application in synergism enhanced remediation of polycyclic aromatic hydrocarbons (PAHs)contaminated soil. Finally, these two types of glycolipid biosurfactants and sorbitolderivants nonionic surfactants were used to develop high efficient and low toxic oilspill dispersants. The main results were summarized as follows:
1. A sophorolipid production strain with high yields was screened. The structures andcompositions of the biosurfactant produced by the strain grown on different carbonsources were analysed and physicochemical properties of the biosurfactant were alsostudied intensively. Fermentation medium compositions were optimized for theproduction of sophorolipid.
(1) A surfactant producing strain, named as O-13-1, was screened from oilywastewater and identified as Starmerella bombicola based on18S rDNA sequence.The results of TLC, FT-IR and HPLC-MS/MS showed that the biosurfactant producedby strain O-13-1was sophorolipids mixture. Using different media based on glucoseand either oleic acid, rapeseed oil, cottonseed oil, and frying waste oil, thesophorolipids produced mainly existed in the lactone form with the hydroxyl-fattyacids of (ω-1) hydroxyoleic acid (C18:1), such as17-L-([2'-O-β-D-glucopyranosyl-β-D-glucopyranosyl]oxy)-octadecenoic acid-1',4"-lactone-6',6"-diacetate.
(2) Sophorolipid had high surface activity, such as low surface tension and CMC.Meanwhile, the heat resistance, temperature resistance, and acid and alkali resistanceof sophorolipid were high. The minimum surface tension and CMC of the producedsophorolipids in aqueous solution were found to be37.0mN/m and30mg/L,respectively. Unhindered surface activity of the sophorolipids was found at widerange of pHs (2-10), temperature (heating for2h) and salt concentrations (0-20%).The CMC of lactonic sophorolipid increased with the adding of aliphatic alcohol butdecreased with the adding of NaCl.
(3) The orthogonal experiment was used to optimize fermentation mediumcompositions for sophorolipid-producing strain and the compositions were: glucose60g/L, oleic acid60mL/L, yeast extract8g/L, and urea2g/L. The yields ofsophorolipid production were low when using low-cost fermentative substrates, suchas glucose syrup, molasses, cottonseed oil and frying waste oil. Less expensive sophorolipids were obtained by using combinations of glucose and oleic acid/rapeseedoil, and the cost of carbon source per mass yield of sophorolipid production were11040and11425CNY/t. Therefore, glucose, oleic acid, and rapeseed oil can be usedas preferred carbon source for sophorolipids production by O-13-1strain.
2. Micellar and surface properties of sophorolipids and rhamnolipids mixed systemswere studied by a variety of technologies.
(1) CMC of lactonic sophorolipid (LS) and dirhamnolipid (R2) mixed systemswere determined by steady-state fluorescence spectroscopy. Interaction parameter (β)and free energy were calculated according to Rubingh’s model. In the LS/R2mixedsystems, the experimental CMC values were always lower than that calculatedaccording to Clint’ ideal mixing model (CMCideal). The deviation of experimentalCMC from CMCsidealhad a maximum at the mole fraction of LS0.1, and the CMC ofLS/R2mixed system decreased by61.7%and18.9%compared to single LS and R2,respectively. More negative values of β and thermodynamic parameter (ΔGmicand GE)of mixed systems were found when αLSwere between0.025and0.3. These resultsindicate that strong synergistic interactions between LS and R2occurred in mixedmicelles and the mixed micelles formation is spontaneous process when a smallamount of LS in the LS/R2systems.
(2) In view of the fermentation products of sophorolipids and rhamnolipids arealways their homologous mixtures. Micellar and surface properties of sophorolipidshomologs (SLs) and rhamnolipids homologs (RLs) mixed systems were studied bysurface tension measurement. Results showed that the experimental CMC values areobviously lower than the CMCidealvalues in the SLs/RLs mixed system. β was morenegative when a small amount of SLs was added to RLs solution, indicating that SLsin the SLs/RLs systems promote stronger interaction between SLs and RLs. It is ingood agreement with the results of LS/R2mixed systems. The mole fraction of SLs inthe surface (xSLsσ) and mixed micelle (xSLs) increased with the increased of SLs in thesolution (αSLs) and were always higher than αSLs, indicating that both the surfaceadsorption and mixed micelles are dominated by SLs, and the higher contribution ofSLs to synergism.
(3) Steady-state fluorescence spectroscopy, dynamic light scattering (DLS), andcryogenic transmission electron microscopy (cryo-TEM) results showed that the Naggvalues of LS/R2mixed systems were small but micelle sizes were large. The Naggvalues were from3to6and monodisperse micelle diameters were in the range of5-20nm when the total concentrations of LS and R2were in the range of0.3-0.8mmol/L. Meanwhile, the large size micelle aggregations were found. The micellediameters of LS/R2mixed systems had a maximum as the mole fraction of LS (αLS) inthe LS/R2systems was0.7, and according micelle diameter was about160nm. BothLS and R2consisted dimeric glucose and long-chain aliphatic, enabled the moleculeto cover large space. The large headgroups and long hydrophobic chain of glycolipidsprevented surfactant aggregation. Consequently, the mixed LS/R2systems formedlarge and incompact vesicular micelles aggregations.
3. Facile solubilization and soil washing of polycyclic aromatic hydrocarbons (PAHs)by sophorolipids and rhamnolipids mixed systems (SLs/RLs) were investigated. Thethe effects that might affect solubilization and desorption efficiency, such as pH,salinity, and heavy metal were also investigated.
(1) The mixed SLs/RLs systems were more effective for the solubilization anddesorption of PAHs than single SLs and RLs. The molar solubilization ratio (MSR) ofphenanthrene (Phen) and pyrene (Py) had maximums at the mole fractions of SLs was0.7. The MSR of Phen for SLs/RLs mixed system (αSLs=0.7) were0.274. It is1.6and8.6times as much as that for single SLs and RLs, respectively. For Py, the MSR was0.079and the corresponding multiples were1.80and5.3, respectively. The desorptionpercentage of Phen and Py for SLs/RLs system (αSLs=0.7) had a maximum of71.5%and59.3%with the total glycolipid concentration of4.2mmol/L, respectively. It is39.7%and35.7%higher than SLs, and about33.2%and32.7%higher than RLs,respectively.
(2) The solubilizing capabilities of SLs/RLs mixed system (αSLs=0.7) increasedwith the increase of pH from5.5to8.0and had maximum when the solution had pHvalues between5.5and6.0. The effect of salinity on the desorption of SLs/RLssystem depended on the concentrations of glycolipid and the difference of salinity. For SLs/RLs mixed system, the desorption of Phen and Py from soil were promotedby the adding of NaCl with the concentration of0.01mol/L when the total glycolipidconcentrations were more than4mmol/L. However, when the concentration of NaClincreased to0.05mol/L, the desorption of Phen and Py from soil were inhibited forboth SLs/RLs mixed system and single SLs and RLs. Meanwhile, the salt resistanceof SLs/RLs mixed system was higher than single SLs and RLs. Therefore, mixedSLs/RLs systems improved salt tolerance of SLs and RLs. Heavy metal (Cadmium)has no significant effect on the desorption of PAHs from soil for single SLs, RLs, andmixed SLs/RLs system.
4. SLs/RLs mixed systems were used to prepare oil spill dispersant. High efficient,low toxic, and environmentally friendly oil spill dispersants were developed. Thesuitable application environmental conditions were identified.
(1) To develop more efficient and less toxic oil spill dispersant of concentratedtype, two kinds of glycolipid biosurfactants (sophorolipid and rhamnolipid) and twokinds of sorbitol derivant nonionic surfactant were chosen in this study. Oneoptimized dispersant formulation SRTG-16was identified by uniform design methods.The formulation composition was as follows: sophorolipids/rhamnolipids/sorbitolderivant1/sorbitol derivant2/solvent:6.80/0.83/3.28/39.10/50. The dispersioneffectiveness (DE) of the dispersant was higher than single surfactant. The dispersanthad the ten minute’ DE of46.30%for QHD32-6crude oil.
(2) The dispersant had higher DE when it was used in suitable environmentalconditions. DE of dispersant kept high at the dispersant-to-oil ratio from1:10to1:25.The maximun DE of56.65%was found at5℃. pH and salinity had no significantinfluence on DE of dispersant. It can be concluded that the dispersant is suitable forremediation of oil spill not only in fresh water but also in seawater.
(3) Danio rerio (freshwater fish) and Tridentiger trigonocephalus (saltwater fish)were used to study the acute toxicity of dispersant SRTG-16. Results showed thatlethality rate for Danio rerio at the end of48hours was0%, and for Tridentigertrigonocephalus, lethality rate was also0%at the end of24hours. It is far better thanthe demand of national standard (GB18181.1-2000). The results showed that the dispersant had lower toxicity.
(4) For the dispersant, the ratio of BOD/COD was33.8%, meeting the demand ofnational standard (GB18181.1-2000), in which this value is greater than30%. Theresult showed that the oil spill dispersant was biodegradable.
The results above showed that the dispersant obtained could be used in oil spillresponse operations under appropriate conditions.
本论文在选育槐糖脂生物表面活性剂高产菌和确定槐糖脂主要理化性质的基础上，综合应用稳态荧光探针法、表面张力法、动态激光散射法和低温透射电镜等方法，系统研究了非离子生物表面活性剂槐糖脂与阴离子生物表面活性剂鼠李糖脂复配体系的胶束形态、胶束结构及热力学和动力学性质，进而探讨了该复配体系对多环芳烃（PAHs）污染土壤增效修复作用及其机制，并应用均匀设计法，开发出以糖脂类生物表面活性剂和非离子的山梨醇酯类表面活性剂复配体系为主体的环境友好型溢油分散剂。论文主要研究结果可归纳如下：
1.筛选出槐糖脂生物表面活性剂高产菌，确定了该菌株利用不同碳源所产槐糖脂同系物的组成和主要理化性质，优化了该菌株利用不同底物发酵制备槐糖脂的培养基组成。
（1）从含油废水中筛选出生物表面活性剂高产菌O-13-1，经18S rDNA测序鉴定为假丝酵母菌（Starmerella bombicola），所产生物表面活性剂经薄层层析（TLC）、红外光谱（FT-IR）及液质联用（HPLC-MS/MS）确定为槐糖脂同系物。该菌株以葡萄糖为水溶性底物和以油酸、菜籽油、棉籽油和煎炸废油等脂溶性底物为双碳源发酵时，产物均为含多种自由酸型和内酯型槐糖脂同系物，而内酯型同系物为主要成分，在槐糖内酯中均以脂肪烃链为单不饱和的十八烯酸（C18:1）结构为主，如ω-1型17-L-[(2'-O-β-D-吡喃葡萄糖基-β-D-吡喃葡萄糖基)-O-]-十八烯酸-1',4"-内酯-6',6"二乙酸酯。
（2）槐糖脂溶液具有良好的表面活性、较低的临界胶束浓度（CMC）和良好的耐温性、耐酸碱性和热稳定性。该糖脂溶液能使水的表面张力将至37.0mN/m，CMC仅为30mg/L，在100℃下加热2h、NaCl浓度为0-20%和pH为2-10范围时均保持较高表面活性；而槐糖内脂的CMC值随短链脂肪醇的加入而增大，随NaCl电解质的加入而减小。
（3）通过设计正交试验，优化确定O-13-1菌株产槐糖脂的摇瓶发酵培养基组成为：葡萄糖60g/L；油酸60mL/L，酵母粉8g/L，尿素2g/L。而该菌株利用廉价水溶性碳源如葡萄糖浆和糖蜜以及脂溶性碳源如棉籽油和煎炸废油时槐糖脂产量较低；而以葡萄糖和油酸或葡萄糖和菜籽油作为双碳源时，发酵制备槐糖脂的碳源成本较低，分别为11040元/吨和11425元/吨。因此，O-13-1菌株发酵合成槐糖脂的优选水溶性碳源为葡萄糖，优选脂溶性碳源为油酸和菜籽油。
2.综合应用多种技术手段研究了槐糖脂和鼠李糖脂复配体系混合胶束和表面吸附性质。
（1）首先，采用稳态荧光探针方法测定了非离子生物表面活性剂槐糖内酯（LS）单组分和阴离子生物表面活性剂双鼠李糖脂（R2）单组分按比例复配所形成的混合胶束的CMC，并通过Rubingh规则溶液理论模型计算了二者相互作用参数β及热力学函数。LS/R2混合体系的CMC低于根据Clint理论混合模型计算的CMCideal值，在LS摩尔分数（αLS）=0.1时，该复配体系的协同增效作用最为明显，混合体系的CMC值较单一LS或R2体系的CMC值分别降低了61.7%和18.9%。αLS在0.025-0.3之间时，复配体系胶束相互作用参数β以及混合胶束形成自由能ΔGmic和超额自由能GE值更负，说明在溶液中有少量LS存在时，LS和R2在胶束中有强烈的吸引作用，易于自发形成混合胶束。
（2）鉴于实际发酵得到的槐糖脂和鼠李糖脂都为含有多种组分的同系物，本文进一步采用表面张力方法研究了槐糖脂同系物（SLs）和鼠李糖脂同系物（RLs）复配体系特点，通过吉布斯公式和Rubingh规则溶液理论模型计算了二者在表面吸附层和胶束中行为。结果显示复配体系的CMC实验值低于CMCideal，当溶液中含有少量的槐糖脂时，β更负，SLs和RLs在混合胶束中表现出强烈的吸引作用，与二者单体复配体系研究结果一致。槐糖脂在表面吸附层中的摩尔分数（xSLsσ）及混合胶束中的摩尔分数（xSLs）随其在溶液中摩尔分数（αSLs）的增大而增大且均大于αSLs，槐糖脂在表面吸附层和混合胶束中占优，对复配增效作用贡献较大。
（3）稳态荧光探针方法、动态激光散射法和低温透射电镜测定结果表明，在两种糖脂总浓度为0.3-0.8mmol/L范围时，SL/R2复配体系单分散胶束的Nagg仅为3-6，平均胶束直径约5-20nm，SL/R2复配体系所形成胶束具有聚集数（Nagg）小而胶束直径大的特点。同时在糖脂体系中，单分散胶束发生聚集形成胶束聚集体，当LS摩尔分数为0.7时，胶束聚集体直径最大，达160nm。这是由于槐糖内脂和双鼠李糖脂分子中均含有葡萄糖基和长链疏水基，占据较大空间，不利于分子间的有序排列，导致其复配体系形成尺寸较大的囊泡状胶束聚集体结构，可能对疏水性有机物具有较强增溶能力。
3.研究了SLs/RLs复配体系对水溶液中PAHs的增溶作用及其对PAHs污染土壤的淋洗修复作用，并考察了pH值、盐度和重金属离子等对SLs/RLs复配体系增效修复PAHs污染土壤的影响。
（1）SLs/RLs复配体系对水溶液中PAHs的增溶作用和对土壤中PAHs的洗脱作用明显优于二者单一存在的体系，且对于疏水性相对较弱的菲的增溶和解吸能力大于疏水性较强的芘。如在SLs摩尔分数为0.7时，SLs/RLs复配体系对PAHs的协同增溶作用最强，对菲的摩尔增溶比（MSR）为0.274，分别是SLs和RLs单一体系的1.6倍和8.6倍，对芘的MSR为0.079，分别为SLs和RLs单一体系的1.8和5.3倍。2种糖脂总浓度为4.2mmol/L且SLs摩尔分数为0.7时，SLs/RLs复配体系协同去除菲和芘的效率最高，分别达到71.5%和59.3%，较单一的SLs体系分别高出39.7%和35.7%，而较单一的RLs体系分别高出33.2%和32.7%。
（2）溶液pH值对SLs/RLs复配体系增溶PAHs能力有显著影响，在pH为5.5-8.0的范围内，其增溶能力随pH值的增大而减小，在pH为5.5时，SLs/RLs形成的混合胶束对PAHs的增溶效率最高。盐度对SLs/RLs复配体系洗脱PAHs作用的影响与表面活性剂浓度大小和盐度大小密切相关。两种糖脂总浓度小于3mmol/L时，洗脱液中添加0.01mol/L NaCl对SLs/RLs复配体系洗脱PAHs具有抑制作用，而两种糖脂总浓度大于4mmol/L时，洗脱液中添加0.01mol/L NaCl则对SLs/RLs复配体系洗脱PAHs具有促进作用；当洗脱液中添加0.05mol/LNaCl时，对SLs/RLs复配体系洗脱PAHs都具有抑制作用，但抑制作用明显小于单一的槐糖脂和鼠李糖脂体系，表明SLs/RLs复配体系具有更强的耐盐性；而共存重金属Cd2+对于槐糖脂、鼠李糖脂及复配体系解吸PAHs能力的影响较小。
4.将槐糖脂和鼠李糖脂复配体系应用到溢油分散剂制备中，开发出高效低毒的环境友好型溢油分散剂，并确定了该溢油分散剂的适宜应用条件。
（1）应用均匀实验设计方法，以分散率为主要评价指标，确定了以槐糖脂、鼠李糖脂、以及两种山梨醇酯类非离子表面活性剂复配体系为主体的浓缩型溢油分散剂配方SRTG-16。该配方为槐糖脂/鼠李糖脂/山梨醇酯1/山梨醇酯2/溶剂：6.80/0.83/3.28/39.10/50，该溢油分散剂对QHD32-6原油的分散性能优于任一单一表面活性剂，10min分散率达到46.30%。
（2）确定了该溢油分散剂的适宜应用条件为：剂油比，1:10-1:25；温度，5-20℃；盐度，0-40；pH，6.0-10.0。
（3）分别以斑马鱼（Danio rerio）和纹缟鰕虎鱼（Tridentiger trigonocephalus）为受试生物，通过急性毒性试验确定所开发的溢油分散剂SRTG-16的生物毒性，结果表明该溢油分散剂对斑马鱼（D. rerio）的48h致死率和对纹缟鰕虎鱼（T.trigonocephalus）的24h致死率都为0%，明显优于国标（GB18181.1-2000）中半致死时间大于24h的指标，表明该溢油分散剂具有较低的生物毒性。
（4）该溢油分散剂的BOD/COD值为33.8%，满足国标（GB18181.1-2000）中大于30%的要求，表明该溢油分散剂具有良好的生物可降解性。
上述结果表明所开发的溢油分散剂适用于海面溢油污染修复和治理。
Surfactant enhanced remediation (SEM) has been developed to be a promisingremediation method for the removal of hydrophobic organic compounds (HOCs) fromgroundwater and soil. Recently, the use of a wide range of surfactants in SEMremediation technology are synthetic surfactants. Biosurfactants have gained growinginterest due to their distinct advantages over their synthetic counterparts, such asspecificity, biodegradability, nontoxicity, and a broad range of structures. Mixedsurfactant systems often present superior properties to those with a single surfactantcomponent, such as better efficiency in decreasing surface/interfacial tensions, lowercritical micelle concentration (CMC), higher solubilizing power, and expandedcolloidal stability. Therefore, mixed surfactant systems, especially biosurfactantmixed systems, have good prospects of application for the remediation ofhydrophobic organics contaminated site. However, few recent studies havehighlighted the micelle structure and properties of biosurfactants, especially mixedbiosurfactants. The limited number of works involving these aspects has prevented adeeper knowledge and understanding of biosurfactants application insurfactant-enhanced remediation of hydrophobic organics contaminated. Therefore, itis difficult to guide the application of biosurfactants and mixed bisurfactant forremediation of hydrophobic organics contaminated site.
In this study, sophorolipid production strain with high yields was first screenedand main physicochemical properties were determined. Micellar and surfaceproperties of sophorolipid and rhamnolipid mixed systems were studied bysteady-state fluorescence spectroscopy, surface tension measurements, dynamic lightscattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM). Then,sophorolipids and rhamnolipids mixed systems were investigated for their application in synergism enhanced remediation of polycyclic aromatic hydrocarbons (PAHs)contaminated soil. Finally, these two types of glycolipid biosurfactants and sorbitolderivants nonionic surfactants were used to develop high efficient and low toxic oilspill dispersants. The main results were summarized as follows:
1. A sophorolipid production strain with high yields was screened. The structures andcompositions of the biosurfactant produced by the strain grown on different carbonsources were analysed and physicochemical properties of the biosurfactant were alsostudied intensively. Fermentation medium compositions were optimized for theproduction of sophorolipid.
(1) A surfactant producing strain, named as O-13-1, was screened from oilywastewater and identified as Starmerella bombicola based on18S rDNA sequence.The results of TLC, FT-IR and HPLC-MS/MS showed that the biosurfactant producedby strain O-13-1was sophorolipids mixture. Using different media based on glucoseand either oleic acid, rapeseed oil, cottonseed oil, and frying waste oil, thesophorolipids produced mainly existed in the lactone form with the hydroxyl-fattyacids of (ω-1) hydroxyoleic acid (C18:1), such as17-L-([2'-O-β-D-glucopyranosyl-β-D-glucopyranosyl]oxy)-octadecenoic acid-1',4"-lactone-6',6"-diacetate.
(2) Sophorolipid had high surface activity, such as low surface tension and CMC.Meanwhile, the heat resistance, temperature resistance, and acid and alkali resistanceof sophorolipid were high. The minimum surface tension and CMC of the producedsophorolipids in aqueous solution were found to be37.0mN/m and30mg/L,respectively. Unhindered surface activity of the sophorolipids was found at widerange of pHs (2-10), temperature (heating for2h) and salt concentrations (0-20%).The CMC of lactonic sophorolipid increased with the adding of aliphatic alcohol butdecreased with the adding of NaCl.
(3) The orthogonal experiment was used to optimize fermentation mediumcompositions for sophorolipid-producing strain and the compositions were: glucose60g/L, oleic acid60mL/L, yeast extract8g/L, and urea2g/L. The yields ofsophorolipid production were low when using low-cost fermentative substrates, suchas glucose syrup, molasses, cottonseed oil and frying waste oil. Less expensive sophorolipids were obtained by using combinations of glucose and oleic acid/rapeseedoil, and the cost of carbon source per mass yield of sophorolipid production were11040and11425CNY/t. Therefore, glucose, oleic acid, and rapeseed oil can be usedas preferred carbon source for sophorolipids production by O-13-1strain.
2. Micellar and surface properties of sophorolipids and rhamnolipids mixed systemswere studied by a variety of technologies.
(1) CMC of lactonic sophorolipid (LS) and dirhamnolipid (R2) mixed systemswere determined by steady-state fluorescence spectroscopy. Interaction parameter (β)and free energy were calculated according to Rubingh’s model. In the LS/R2mixedsystems, the experimental CMC values were always lower than that calculatedaccording to Clint’ ideal mixing model (CMCideal). The deviation of experimentalCMC from CMCsidealhad a maximum at the mole fraction of LS0.1, and the CMC ofLS/R2mixed system decreased by61.7%and18.9%compared to single LS and R2,respectively. More negative values of β and thermodynamic parameter (ΔGmicand GE)of mixed systems were found when αLSwere between0.025and0.3. These resultsindicate that strong synergistic interactions between LS and R2occurred in mixedmicelles and the mixed micelles formation is spontaneous process when a smallamount of LS in the LS/R2systems.
(2) In view of the fermentation products of sophorolipids and rhamnolipids arealways their homologous mixtures. Micellar and surface properties of sophorolipidshomologs (SLs) and rhamnolipids homologs (RLs) mixed systems were studied bysurface tension measurement. Results showed that the experimental CMC values areobviously lower than the CMCidealvalues in the SLs/RLs mixed system. β was morenegative when a small amount of SLs was added to RLs solution, indicating that SLsin the SLs/RLs systems promote stronger interaction between SLs and RLs. It is ingood agreement with the results of LS/R2mixed systems. The mole fraction of SLs inthe surface (xSLsσ) and mixed micelle (xSLs) increased with the increased of SLs in thesolution (αSLs) and were always higher than αSLs, indicating that both the surfaceadsorption and mixed micelles are dominated by SLs, and the higher contribution ofSLs to synergism.
(3) Steady-state fluorescence spectroscopy, dynamic light scattering (DLS), andcryogenic transmission electron microscopy (cryo-TEM) results showed that the Naggvalues of LS/R2mixed systems were small but micelle sizes were large. The Naggvalues were from3to6and monodisperse micelle diameters were in the range of5-20nm when the total concentrations of LS and R2were in the range of0.3-0.8mmol/L. Meanwhile, the large size micelle aggregations were found. The micellediameters of LS/R2mixed systems had a maximum as the mole fraction of LS (αLS) inthe LS/R2systems was0.7, and according micelle diameter was about160nm. BothLS and R2consisted dimeric glucose and long-chain aliphatic, enabled the moleculeto cover large space. The large headgroups and long hydrophobic chain of glycolipidsprevented surfactant aggregation. Consequently, the mixed LS/R2systems formedlarge and incompact vesicular micelles aggregations.
3. Facile solubilization and soil washing of polycyclic aromatic hydrocarbons (PAHs)by sophorolipids and rhamnolipids mixed systems (SLs/RLs) were investigated. Thethe effects that might affect solubilization and desorption efficiency, such as pH,salinity, and heavy metal were also investigated.
(1) The mixed SLs/RLs systems were more effective for the solubilization anddesorption of PAHs than single SLs and RLs. The molar solubilization ratio (MSR) ofphenanthrene (Phen) and pyrene (Py) had maximums at the mole fractions of SLs was0.7. The MSR of Phen for SLs/RLs mixed system (αSLs=0.7) were0.274. It is1.6and8.6times as much as that for single SLs and RLs, respectively. For Py, the MSR was0.079and the corresponding multiples were1.80and5.3, respectively. The desorptionpercentage of Phen and Py for SLs/RLs system (αSLs=0.7) had a maximum of71.5%and59.3%with the total glycolipid concentration of4.2mmol/L, respectively. It is39.7%and35.7%higher than SLs, and about33.2%and32.7%higher than RLs,respectively.
(2) The solubilizing capabilities of SLs/RLs mixed system (αSLs=0.7) increasedwith the increase of pH from5.5to8.0and had maximum when the solution had pHvalues between5.5and6.0. The effect of salinity on the desorption of SLs/RLssystem depended on the concentrations of glycolipid and the difference of salinity. For SLs/RLs mixed system, the desorption of Phen and Py from soil were promotedby the adding of NaCl with the concentration of0.01mol/L when the total glycolipidconcentrations were more than4mmol/L. However, when the concentration of NaClincreased to0.05mol/L, the desorption of Phen and Py from soil were inhibited forboth SLs/RLs mixed system and single SLs and RLs. Meanwhile, the salt resistanceof SLs/RLs mixed system was higher than single SLs and RLs. Therefore, mixedSLs/RLs systems improved salt tolerance of SLs and RLs. Heavy metal (Cadmium)has no significant effect on the desorption of PAHs from soil for single SLs, RLs, andmixed SLs/RLs system.
4. SLs/RLs mixed systems were used to prepare oil spill dispersant. High efficient,low toxic, and environmentally friendly oil spill dispersants were developed. Thesuitable application environmental conditions were identified.
(1) To develop more efficient and less toxic oil spill dispersant of concentratedtype, two kinds of glycolipid biosurfactants (sophorolipid and rhamnolipid) and twokinds of sorbitol derivant nonionic surfactant were chosen in this study. Oneoptimized dispersant formulation SRTG-16was identified by uniform design methods.The formulation composition was as follows: sophorolipids/rhamnolipids/sorbitolderivant1/sorbitol derivant2/solvent:6.80/0.83/3.28/39.10/50. The dispersioneffectiveness (DE) of the dispersant was higher than single surfactant. The dispersanthad the ten minute’ DE of46.30%for QHD32-6crude oil.
(2) The dispersant had higher DE when it was used in suitable environmentalconditions. DE of dispersant kept high at the dispersant-to-oil ratio from1:10to1:25.The maximun DE of56.65%was found at5℃. pH and salinity had no significantinfluence on DE of dispersant. It can be concluded that the dispersant is suitable forremediation of oil spill not only in fresh water but also in seawater.
(3) Danio rerio (freshwater fish) and Tridentiger trigonocephalus (saltwater fish)were used to study the acute toxicity of dispersant SRTG-16. Results showed thatlethality rate for Danio rerio at the end of48hours was0%, and for Tridentigertrigonocephalus, lethality rate was also0%at the end of24hours. It is far better thanthe demand of national standard (GB18181.1-2000). The results showed that the dispersant had lower toxicity.
(4) For the dispersant, the ratio of BOD/COD was33.8%, meeting the demand ofnational standard (GB18181.1-2000), in which this value is greater than30%. Theresult showed that the oil spill dispersant was biodegradable.
The results above showed that the dispersant obtained could be used in oil spillresponse operations under appropriate conditions.
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