戈登氏菌Gordonia sp.WQ-01对石油中二苯并噻吩（DBT）生物脱硫的研究
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摘要
本实验室从大港油田污水中筛选分离出一株能够以DBT为硫源的专一性脱硫菌株WQ-01。色谱分析表明，WQ-01以“4S”途径脱除DBT中的硫，并且生成产物二羟基联苯（2-HBP）。由于WQ-01能够通过“4S”途径专一地作用于DBT的C-S键，而不影响C-C键，大大保留了石油燃料原有的燃烧值。
通过形态特征和生理生化特征，并结合16SrRNA分析，从分子水平上确定该菌种为戈登氏菌，命名为Gordonia sp. WQ-01。
为进一步提高WQ-01的脱硫能力，本课题使用了激光诱变育种技术。研究了不同照射功率、不同照射剂量对细胞的存活率和正突变率的影响。同时还以WQ-01与其激光诱变菌WQ-01A为研究对象，从温度效应、通透性变化、酶活及其编码酶基因序列、蛋白质结构等方面考察了激光诱变对戈登氏菌脱硫活性的影响。结果表明：温度效应和通透性并不是诱变菌株脱硫活性提高的主要原因；由基因的序列改变引发的酶的氨基酸序列，二级结构和空间结构发生的变化才是脱硫活性提高的根本原因。
以突变株Gordonia sp. WQ-01A为研究对象进行固定化研究，考察了其静止细胞的最佳固定化条件，对比了水相中固定化细胞和静止细胞的脱硫性能。研究确定了Gordonia sp. WQ-01A最佳固定化条件：菌株经二次活化30℃摇床200rpm培养36小时后制备静止细胞；固定化载体海藻酸钠（SA）质量分数4％（w/v）；菌体质量（g）与胶液体积（mL）比为1:20；4℃下在3％（w/v）氯化钙溶液中充分交联。同时，选取正十二烷作为模拟油相，固定化凝胶小球作为催化剂进行脱硫实验，考察油－水－固定化三相体系中固定化Gordonia sp. WQ-01A静止细胞降解DBT的能力。基于对该体系固定化静止细胞降解DBT反应过程的分析，本文建立一个模拟固定化静止细胞脱硫过程的数学模型研究其动力学行为。模型综合考虑了DBT和溶氧在凝胶小球内、外的传质阻力及DBT－溶氧双底物本征动力学，通过比较模型模拟与油相中实际测得DBT浓度的实验数据验证了该模型的合理性和可靠性。此外，模型对凝胶小球内DBT、溶氧随时间和半径的变化关系做了合理的预测分析。
A strain WQ-01was isolated from polluted water of Dagang Oil-field and it iscapable of specifically desulfurizing DBT as its sulfur source. HPLC shows the strainWQ-01can remove sulfur from DBT, yielding2-HBP through “4S” pathway, bywhich breaks the C-S-bond but remains the C-C-bond so that it can greatly keep thecaloric value of fuel.
According to the morphologic properties, taxonomic properties and the16SrRNAanalysis of the strain, we identified it as Gordonia, named Gordonia sp. WQ-01.
Laser was employed to irradiate the cells of the strain WQ-01to obtain themutant with the higher ability to desulfurize DBT. The effect of He-Ne laserirradiation on the induction of biodesulfuring activity and surviving fraction of cellsof Gordonia sp. WQ-01was studied. With WQ-01and laser-induced mutation strainWQ-01A, we studied the effects of laser-induced mutation on desulfurization of theGordonia sp. WQ-01by checking temperature effect, the permeability of cell, theactivity of enzyme related to desulfurization, the sequences of genes coded enzymeand the structures of enzyme. Based on the above analysis, it can be draw theconclusion that the improved desulfurizing activity of the mutant Gordonia sp.WQ-01was due to the increased activity of the enzymes involved in the “4S” pathway.The essential reason is the change of the sequences of the genes coding desulfurizingenzymes, not the temperature effect or the change of permeability of cell.
At last, immobilization of Gordonia sp. WQ-01A resting cells was studied. Theeffects of immobilization conditions on biodesulfurization were investigated.Microstructure of alginate gel beads was observed by SEM and the biodesulfurizationcharacteristics between immobilized cells and resting cells was compared in waterphase. The results showed that the optimum immobilization condition is: bacteria iscultivated at30℃for36hours after activation twice; the concentration of carrier,sodium alginate (SA), is4%(w/v) and the ratio of cells (g) to SA (mL) is1:20withthe3%(w/v)calcium chloride as the precipitator under4℃.
Batch DBT biodesulfurization in the oil-water-immobilization system wasconducted. A mathematical model was developed to simulate the biodesulfurizationprocess, which took into account the internal and external mass transfer resistances of DBT and oxygen and the intrinsic kinetics of bacteria. The good agreement betweenthe model simulations and the experimental measurements of DBT concentrationprofiles validated the proposed model. Moreover, the time and radius courses of DBTand oxygen concentration profiles within the alginate gel beads were reasonablypredicted and analysed by the proposed model.
通过形态特征和生理生化特征，并结合16SrRNA分析，从分子水平上确定该菌种为戈登氏菌，命名为Gordonia sp. WQ-01。
为进一步提高WQ-01的脱硫能力，本课题使用了激光诱变育种技术。研究了不同照射功率、不同照射剂量对细胞的存活率和正突变率的影响。同时还以WQ-01与其激光诱变菌WQ-01A为研究对象，从温度效应、通透性变化、酶活及其编码酶基因序列、蛋白质结构等方面考察了激光诱变对戈登氏菌脱硫活性的影响。结果表明：温度效应和通透性并不是诱变菌株脱硫活性提高的主要原因；由基因的序列改变引发的酶的氨基酸序列，二级结构和空间结构发生的变化才是脱硫活性提高的根本原因。
以突变株Gordonia sp. WQ-01A为研究对象进行固定化研究，考察了其静止细胞的最佳固定化条件，对比了水相中固定化细胞和静止细胞的脱硫性能。研究确定了Gordonia sp. WQ-01A最佳固定化条件：菌株经二次活化30℃摇床200rpm培养36小时后制备静止细胞；固定化载体海藻酸钠（SA）质量分数4％（w/v）；菌体质量（g）与胶液体积（mL）比为1:20；4℃下在3％（w/v）氯化钙溶液中充分交联。同时，选取正十二烷作为模拟油相，固定化凝胶小球作为催化剂进行脱硫实验，考察油－水－固定化三相体系中固定化Gordonia sp. WQ-01A静止细胞降解DBT的能力。基于对该体系固定化静止细胞降解DBT反应过程的分析，本文建立一个模拟固定化静止细胞脱硫过程的数学模型研究其动力学行为。模型综合考虑了DBT和溶氧在凝胶小球内、外的传质阻力及DBT－溶氧双底物本征动力学，通过比较模型模拟与油相中实际测得DBT浓度的实验数据验证了该模型的合理性和可靠性。此外，模型对凝胶小球内DBT、溶氧随时间和半径的变化关系做了合理的预测分析。
A strain WQ-01was isolated from polluted water of Dagang Oil-field and it iscapable of specifically desulfurizing DBT as its sulfur source. HPLC shows the strainWQ-01can remove sulfur from DBT, yielding2-HBP through “4S” pathway, bywhich breaks the C-S-bond but remains the C-C-bond so that it can greatly keep thecaloric value of fuel.
According to the morphologic properties, taxonomic properties and the16SrRNAanalysis of the strain, we identified it as Gordonia, named Gordonia sp. WQ-01.
Laser was employed to irradiate the cells of the strain WQ-01to obtain themutant with the higher ability to desulfurize DBT. The effect of He-Ne laserirradiation on the induction of biodesulfuring activity and surviving fraction of cellsof Gordonia sp. WQ-01was studied. With WQ-01and laser-induced mutation strainWQ-01A, we studied the effects of laser-induced mutation on desulfurization of theGordonia sp. WQ-01by checking temperature effect, the permeability of cell, theactivity of enzyme related to desulfurization, the sequences of genes coded enzymeand the structures of enzyme. Based on the above analysis, it can be draw theconclusion that the improved desulfurizing activity of the mutant Gordonia sp.WQ-01was due to the increased activity of the enzymes involved in the “4S” pathway.The essential reason is the change of the sequences of the genes coding desulfurizingenzymes, not the temperature effect or the change of permeability of cell.
At last, immobilization of Gordonia sp. WQ-01A resting cells was studied. Theeffects of immobilization conditions on biodesulfurization were investigated.Microstructure of alginate gel beads was observed by SEM and the biodesulfurizationcharacteristics between immobilized cells and resting cells was compared in waterphase. The results showed that the optimum immobilization condition is: bacteria iscultivated at30℃for36hours after activation twice; the concentration of carrier,sodium alginate (SA), is4%(w/v) and the ratio of cells (g) to SA (mL) is1:20withthe3%(w/v)calcium chloride as the precipitator under4℃.
Batch DBT biodesulfurization in the oil-water-immobilization system wasconducted. A mathematical model was developed to simulate the biodesulfurizationprocess, which took into account the internal and external mass transfer resistances of DBT and oxygen and the intrinsic kinetics of bacteria. The good agreement betweenthe model simulations and the experimental measurements of DBT concentrationprofiles validated the proposed model. Moreover, the time and radius courses of DBTand oxygen concentration profiles within the alginate gel beads were reasonablypredicted and analysed by the proposed model.
引文
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