本研究以大港油田落地油污染土壤中筛选出的能高效降解石油烃类物质的假单胞菌DG17为靶微生物，以原油和正十八烷为代表性目标污染物，研究了假单胞菌对原油中不同组分的假增溶能力，并深入分析了微生物对正十八烷的吸附摄取方式，继而探索假单胞菌DG17对正十八烷的跨膜运输方式以及包气带环境因素变化对正十八烷跨膜运输的影响。寻找影响和限制微生物降解烷烃类物质的关键环节和控速步骤可为生物修复石油污染土壤提供一定理论支撑。在以原油为生物利用碳源的摇瓶培养过程中，部分原油组分从油膜中在假单胞菌DG17作用下进入到水相中。GC-MS分析结果表明这些优先被微生物乳化的烃类物质主要为C12 到 C30 的正烷烃类物质，且发酵液表面张力降低。通过相差显微镜观察，发现这些石油烃类物质可悬浮在水相中，且油滴大小随着微生物的生长逐渐变小。在200 mg•L-1和400 mg•L-1正十八烷条件下，假单胞菌DG17生长良好，发酵液表面张力明显降低，生物表面活性剂的生长量也相对较高。而当底物浓度低于100 mg•L-1时，微生物DG17生长反而受到抑制。电喷雾-质谱结果表明DG17分泌的生物表面活性剂主要为鼠李糖脂类物质。在以上研究的基础上，提出实验所用靶微生物假单胞菌对石油烃类物质的吸附概念模型，即假单胞菌DG17首先要直接接触油水界面处的烃类物质，这种情况下的吸附面积相对较低。然后再通过分泌生物表面活性剂方式吸附乳化的或者假增溶的烃类物质油滴，此时微生物和烃类油滴的吸附面积大大增加。因此，在微生物—石油烃类物质体系中，微生物与疏水性烃类物质的吸附是影响石油烃类物质生物降解的关键环节和控速步骤之一。利用同位素[14C]示踪技术分析假单胞菌DG17对正十八烷的跨膜运输方式，发现底物浓度为9.09—36.36µmol•L-1时，膜外[14C]正十八烷可能通过被动运输的方式跨膜进入DG17膜内，且膜内富集[14C]正十八烷的量可达到一个饱和值。但当低[14C]正十八烷浓度为2.227—4.54µmol•L-1时，能量抑制剂会明显抑制DG17跨膜摄取[14C]正十八烷，DG17可能通过与能量有关的主动运输方式跨膜运输膜外碳源。通过米氏方程拟合，得到假单胞菌DG17对[14C]正十八烷的最大降解速率[14C] Vmax为0.13μmol•L-1•h-1，降解速率达到最大速率一半时的底物浓度Kt为11.70μmol•L-1。DG17对[14C]正十八烷的最大吸附摄取速率[14C] Vmax为0.96 μmol•min-1•mg-1 protein，吸附摄取速率达到最大速率一半时的底物浓度Kt为11.27μmol•L-1。对不同包气带环境因素下假单胞菌DG17对[14C]正十八烷的跨膜运输进行分析，发现底物类似物、pH和盐度对假单胞菌DG17跨膜运输膜外[14C]正十八烷有明显影响。偏酸偏碱和高盐度环境可通过破坏假单胞菌DG17细胞膜结构以及膜内外渗透压进而抑制其对膜外烷烃的跨膜摄取；底物类似物作为竞争性物质同样可被DG17跨膜运输从而抑制正十八烷的跨膜摄取；而缺乏营养元素N和P对DG17跨膜运输膜外[14C]正十八烷没有明显影响，但是会抑制微生物的生长繁殖进而降低假单胞菌DG17对[14C]正十八烷的生物降解效率。

In the bioremediation process of hydrocarbons polluted soil, microorganisms are considered to be the major role to biodegrade these hydrocarbons. Thus, it has widely focuses on the uptake and trans-membrane transport mechanism of hydrocarbons. In this study, the pseudosolubiliz ability of Pseudomonas sp.DG17 on curde oil and other alkanes, uptake and transport mechanisms of n-octadecane were analyzed, as well as environmental factors on the transport of n-octadecane by Pseudomonas sp.DG17. All of these studies were aim to implore the key factors that influence biodegradation of hydrocarbons and apply some theories for bioremediation of petroleum polluted soils.Durying incubation time, culture medium turned into brown which indicated that some oil components were transferred into water phase under the effect of Pseudomonas sp.DG17. GC-MS results showed that pseudosolubilized oil components were mainly C12 to C30 of n-alkanes. Furtherore, surface tension of medium also decreased which showed that biosurfactants were secreted by Pseudomonas sp DG17. Phase-contrast micrograph observation directly showed that some crude oil components entered into water phase. Size of these oil droples decreased along with the growh of bacteria.Culture growth result showed that Pseudomonas sp. DG17 grew well in the addition of 200 and 400 mg•L-1 of octadecane. Furthermore, surface tension decreased obviously and content of biosurfactants were high at high substrate concentrations. However, growh Pseudomonas sp. DG17 seems inhibited at lower substrate concentrations. EIS-MS results showed that Pseudomonas sp. DG17 produced rhamnolipids biosurfactant that contains 10 congeners, including monorhamnolipids and dirhamnolipids. These results indicated that the direct contact with insoluble octadecane droplets occurred before the contact with emulsified smaller oil droplets. In this base, the modle of hydrocarbon uptake by microorganisms was as follows: adsorbtion area between microorganism and oil droplets was limited in the environment. However, functional microorganisms could adsorb pseudosolubilized small oil droplets by produing biosurfactants, which could lower surface tension between oil and water interface. Thus, uptake of hydrophobic hydrocarbons was a key factor that influenced the biodegradation process of these compounds.Trans-membrane transport of [14C] n-octadecane by Pseudomonas sp. DG17 was studied by using isotope tracing technique. Cellular and extracellular [14C] n-octadecane of Pseudomonas sp. DG17 were analyzed simultaneously. The results showed that n-octadecane was transported intracellular through passive diffusion at high substrate concentrations, and achieved at steady level. However, transport of n-octadecane was also observed against concentration gradient at lower substrate concentrations for ATP inhibitors decreased cellular n-octadecane a lot. This result revealed that an energy-dependent active transport exhibited in n-octadecane influx. Furthermore, Pseudomonas sp. DG17 could biodegrade n-octadecane well with a Kt value of 11.70µmol•L-1 and a Vmax value of 0.13 µmol•L-1•h-1 and initial uptake of [14C] was specific with an apparent dissociation constant (Kt) of 11.27µmol•L-1 and Vmax of 0.96µmol•min-1•mg-1 protein. Effect of different environmental factors, including pH, salt concentration, substrate analogues and nutrients, on the transport of [14C] n-octadecane by Pseudomonas sp. DG17 were also studied. Results showed that high salt concentrations, low pH values and high pH values could obviously inhibit transportation of extracellular [14C] n-octadecane. Cell membrane and osmotic balance of microorganism might be destroyed which directly cased transportation of hydrocarbons inhibit. On the other hand, deficiency of nutrient, including nitrogen and phosphorus, could not support the growth of biodegradable microorganism, and then lowered biodegradability of [14C] n-octadecane by Pseudomonas sp. DG17. However, this extreme environment seemed not influence tras-membrane transport of [14C] n-octadecane.