海洋近岸溢油污染微生物修复技术的应用基础研究
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摘要
筛选自长期受到石油污染区域的高效石油烃降解菌株，在实验室的研究过程中一般都会表现出较高的烃降解性能，但是当把它们投放到现场的应用环境中后，由于缺乏对现场环境的适应能力，大多数微生物菌株并不能很好地再现其高效的石油烃降解性能。国外对微生物修复处理技术的应用研究较早，而在国内，微生物修复的处理技术还处在实验室的研究阶段，很少有大规模现场实验的开展，更无严格的应用标准进行遵循。因此，本论文通过从实验室研究到现场应用的一系列完整的微生物修复实验，探讨了微生物修复技术在海洋近岸溢油污染环境中应用的可行性，并得出以下结论：
（1）从青岛港近岸的水样中筛选得到4株石油烃降解菌: Ochrobactrum sp.N1（No.HQ231209）、Brevibacillus parabrevis N2（No.HQ231210）、Brevibacillusparabrevis N3（No.HQ231211）、Brevibacillus parabrevis N4（No.HQ231212）。将N系列石油烃降解菌和产生物表面活性剂菌株Bbai~(-1)复配成高效石油烃降解菌群，该菌群在富集培养基中经过16个小时进入生长稳定期，在油培养基中经过20小时进入生长稳定期。油培养基中的菌浓可达9.12×10~9cell·mL~(-1)，对原油降解的最适宜环境条件为：pH=8.0，T=25oC，盐度为（NaCl%）25g·L~(-1)。
（2）原油因风化作用在初始阶段的质量损失主要来源于原油中轻质组分的大量挥发，分子量较大且结构复杂的石油烃组分受风化作用的影响较小。通过气相色谱分析，原油在风化过程中的质量损失主要来源于C17之前的烃组分挥发。
通过海水模拟实验柱对油污染水体环境进行了模拟。用化学消油剂处理海水表层的溢油污染物时，随着海水深度的不断增加，海水垂直方向上石油烃组分的浓度逐渐减小；用微生物修复菌剂处理海水中的溢油污染物时，在初始阶段微生物菌株会优先利用溶解态的石油烃组分进行生长代谢，之后在生长过程中产生的生物表面活性剂等代谢产物会促进油组分的溶解，水体表层石油烃浓度高于底层。
在近岸滩涂沉积物模拟实验环境中，通过室内摇瓶实验可知油砂混合物在海水的冲刷作用下经过10小时达到吸附解吸平衡，水体中石油烃组分的含量为6.40mg·L~(-1)。通过柱型模拟实验证明，在近岸滩涂溢油污染区域应加强10cm以内海砂的修复处理工作。
（3）在微生物修复室内摇瓶实验过程中（25oC，120r/min），烃降解菌N1-N4对原油的降解率为63.2%，通过对微生物降解动力学过程的考察，N系列功能菌对原油的降解较好的符合了一级动力学过程。产生物表面活性剂菌株Bbai~(-1)的加入在一定程度上加快了N1-N4对石油烃组分的降解速率，降解率为66%。
（4）微生物修复室内箱体模拟实验是对室内摇瓶实验的放大，在水体温度为20oC时，细菌总数和石油烃降解菌数量维持在10~7CFU mL~(-1)的水平。菌剂对水体表层原油的乳化效果不理想，实验池中溶解态总烃的含量为5.11mg·L~(-1)。在水体温度为25oC时室内温度为19oC，在短时间内细菌总数和石油烃降解菌数量由最初的的10~6～10~7CFU mL~(-1)上升至10~8CFU mL~(-1)，原油的物理状态从实验之初较大的块状逐渐被乳化成很小的球状或片状，水体中溶解态总烃的含量由0.129～0.370mg L~(-1)逐渐上升至32.597～34.366mg L~(-1)，乳化效果优于20oC时的乳化效果。通过GC和GC-MS检测，水体温度为20C，微生物菌剂的降油效果不明显，水体温度为25C，微生物菌剂对石油烃的降解指数可达51.1%。
（5）微生物修复野外模拟实验共进行了103天，实验开始之初对各菌株进行了大型的工业发酵，石油烃降解菌N1-N4系列的菌剂共发酵6吨，产生物表面活性剂菌株Bbai-1发酵4吨，菌剂的保质期为20天。实验池水体中细菌总数和烃类降解菌的数量都经历了先减小后增大而后再减少的过程。添加了微生物修复菌剂的各实验池水体中的细菌总数维持在10~6~10~7CFU·mL~(-1)之间，烃降解菌的数量维持在10~5CFU·mL~(-1)以上，空白池的细菌总数始终在10~4CFU·mL~(-1)左右，石油烃降解菌总数保持在10~3CFU·mL~(-1)以下。总体来看以M06池（油、N1-N4）和M01池（油、N1-N4、Bbai-1、营养盐）中的微生物长势较好。
实验过程中各实验池中细菌所产糖脂含量不断增加，以最佳修复条件下M01池代谢所产糖脂含量最高。通过GC和GC-MS检测微生物代谢所产脂肪酸主要为十六烷酸和二十二烷酸。通过分子生态学的分析可知，随着培养时间的增长，布设了微生物修复菌剂的实验池中条带的亮度和丰度都大幅度上升，变化情况同细菌总数和烃降解菌数量的测定结果相符。通过油指纹分析可知，处于最佳降解条件下的M01池中石油烃降解菌对原油的降解效果最好，平均降解指数达到57.39%，M06、M07和M08池中由于营养盐或表面活性剂的缺乏，外加实验开展期间天气复杂多变，平均降解指数分别为39.88%、30.50%、31.99%。
（6）在微生物修复滩涂现场实验中，利用沸石吸附石油烃降解菌，可以较好地耐受海水的冲刷，从而起到更长久的保菌和修复效果。T04区域细菌总数为5.51×10~7CFU·mL~(-1)，石油烃降解菌数量为1.49×10~6CFU·mL~(-1)。T03区域细菌总数为3.08×106CFU·mL~(-1)，石油烃降解菌数量为5.90×10~5CFU·mL~(-1)。
（7）在微生物修复海底现场实验中，海水表层和底层的各个位点相对于参照位点细菌总数都有不同程度的增长，底层水中的石油烃降解菌的数量要高于表层水中的烃降解菌数量，实验漂移位点的菌数要高于固定位点菌数。各位点微生物所产代谢产物的含量遵循先增加后减少的趋势，漂移位点HD06Wb和HD07Wb所测糖脂含量在第33天达到最高，分别为43.54mg·L~(-1)和42.98mg·L~(-1)，这与菌数变化趋势相对应。分子生态学分析的结果表明第14天和第35天各位点条带的丰度和亮度都比本底值有不同程度的增大，且添加的菌剂的条带在各位点中都有出现且亮度较大。
通过室内摇瓶实验、室内箱体模拟实验、野外模拟实验、滩涂现场实验以及海底现场应用等一系列完整的微生物修复实验，评价和优化了微生物修复技术在实际应用过程中的各项工艺参数，为海洋石油污染微生物修复体系的现场应用提供了技术支持。
Hydrocarbon degrading bacteria are usually screened from oil-contaminatedareas. Generally, they could performe very well in laboratory oil degradingexperiments. But the bacteria usually could not reproduce their efficienthydrocarbon-degrading ability in the field environment due to the lack of the ability toadapt to the on-site envirnment. The study abroad has reached high levels onmicrobial remediation treatment technology while the techonology is still in thelaboratory research stage at home. There are few large-scale field experiments to carryout, even less stringent standards to follow. Experiments were performed to assess theeffect of environmental factors on the viability of oil-degradation bacteria in sea water.The aim is mainly to evaluate the application feasibility and screen the most suitablehydrocarbon-degrading bacteria in a proper way. All the conclusions are as follows:
(1) Four hydrocarbon degrading bacteria were isolated from Qingdao Port:Ochrobactrum sp. N1(No.HQ231209), Brevibacillus parabrevis N2(No.HQ231210),Brevibacillus parabrevis N3(No.HQ231211), Brevibacillus parabrevis N4(No.HQ231212). N series bacteria combined with a biosurfactant producer Bbai~(-1) hashigh efficient in petroleum degrading experiment. The mixed bacteria flora couldreach into the growth stationary phase after16hours culture in the enrichmentmedium. In the oil medium, the bacteria reach into the stationary phase after20hoursculture. Cell concentration was9.12×10~9cell·mL~(-1). The optimum conditons forcrude oil degradation are determined to be pH of8.0, temperature of25oC and salinityof (NaCl%)25g·L~(-1).
(2) The initial stage of quality loss in crude oil weathering process mainlycomes from volatilization of light component. Large molecular weight and structureof the complex hydrocarbon component of oil is rearely affected by weathering.Through gas chromatography analysis of crude oil in the weathering process, lossmainly comes from the hydrocarbon component volatilization before C17.
When the oil spills are treated with chemical dispersants, the oil concentration inseawater decrease along with the increasing of water depth. When micribial agents isapplied in the treatment of oil spills, the bacteria in the initial stage only use thedissoluted oil in seawater. After the bacteria adapt to the marine environment,biosurfactant produced in the growth process could increase the dissolution of the oilcomponent. Hydrocarbon concentration in the surface of the seawater is higher thanthe one in the bottom.
In the oil migration shake flask experiment, the oil-sand mixture reach theadsorption-desorption equilibrium after10hours of washing action of the sea water.The components of petroleum hydrocarbons in water is of6.40mg·L~(-1). Columnarsimulation experiment show that the treatment of inshore oil contaminated areasshould be strengthened within10cm of sea sand.
(3) N series bacteria could degrade63.2%of the crude oil in the shake flaskexperiment. Microbial degradation of crude oil consists with first-order kinetics.Biosurfactant producer strain Bbai-1could accelerate the degradation of crude oil byN series bacteria. The degradation rate is66%.
(4) The indoor mesocosm experiment is the amplification of the shake flaskexperiment. When the temperature is20oC, TVC and the number of the HDB remainat the level of10~7CFU·mL~(-1). The dissolved hydrocarbons concentration is of5.11mg·L~(-1). When the temperature is25oC, the room temperature is19oC. TVC and thenumber of the HDB could reach up to10~8CFU mL~(-1)from10~7CFU mL~(-1). Oilconcentration in seawater is32.597to34.366mg L~(-1). The emulsification efficience isbetter than the one in the temperature of20oC. Based on the GC and GC-MS analysis,the degradation rate is51.1%in the temperature of25oC.
(5) The field simulation experiment is conducted for103days. N series bacteriaagent is fermented a total of six tons. Biosurfactant producer strain Bbai-1isfermented to be4tons. In the experimental pool, TVC and the number of HDB areexperienced a process of decreas, increas and finally decrease. TVC in eachexperimental pool maintain at the level of10~6~10~7CFU·mL~(-1). The number of HDB ismaintained at more than10~5CFU·mL~(-1). TVC and HDB in the control were 10~4CFU·mL~(-1)and10~3CFU·mL~(-1)respectively. The strains in M06(oil, the microbesare growing well in the N1-N4) and M01(oil, N1-N4, Bbai-1nutrient) grow betterthan other pools.The glycolipid content produced by the strains maintains increase ineach experimental pool. Glycolipid in M01has the hightest yield. Based on the GCand GC-MS analysis, the metabolites are mainly hexadecanoic acid and behenic acid.Molecular Ecology's analysis shows that, with the growth of the culture time,brightness and abundance of the inocula experiment pool bands increasedsignificantly. The degradation rate in M01pool was57.39%. The average degradationrates in M06, M07and M08are39.88%,30.50%,31.99%due to the lack of nutrientsor surfactant.
(6)In the bioremediation of shore site experiment, the use of zeolite adsorbinghydrocarbon degrading bacteria could tolerate the washing effect from seawater. Itcan keep the number of function bacteria in a high level. TVC and the number of theHDB in T04region are5.51×10~7CFU·mL~(-1)and1.49×10~6CFU·mL~(-1)respectively.TVC and the number of the HDB in T03region are3.08×10~6CFU·mL~(-1)and5.90×10~5CFU·mL~(-1)respectively.
(7) In the bioremediation of marine sediment experiment, TVC both in thesurface water and in the bottom increase to a certaine extent compared with thecontrol station. The number of HDB in the bottom water is higher than the one in thesurface wate. The bacteria in drifting station are more than the fixed station.
Glycolipid produed in the drifting stations HD06Wb and HD07Wb are43.54mg·L~(-1)and42.98mg·L~(-1)respectively after33days experiment. It is correspondingwith the bacteria growth trend. Molecular ecology analysis results show that theabundance and brightness on the14d and the35d are greater than the background.
Each technological parameter is optimized through the series of bioremediationexperiment from indoor shake flask experiment to marine site application. Theprogram conducted in this study is the exploration, in China, in forming a commonmethod to evaluate petroleum-utilizing abilities of bioremediation agents.
（1）从青岛港近岸的水样中筛选得到4株石油烃降解菌: Ochrobactrum sp.N1（No.HQ231209）、Brevibacillus parabrevis N2（No.HQ231210）、Brevibacillusparabrevis N3（No.HQ231211）、Brevibacillus parabrevis N4（No.HQ231212）。将N系列石油烃降解菌和产生物表面活性剂菌株Bbai~(-1)复配成高效石油烃降解菌群，该菌群在富集培养基中经过16个小时进入生长稳定期，在油培养基中经过20小时进入生长稳定期。油培养基中的菌浓可达9.12×10~9cell·mL~(-1)，对原油降解的最适宜环境条件为：pH=8.0，T=25oC，盐度为（NaCl%）25g·L~(-1)。
（2）原油因风化作用在初始阶段的质量损失主要来源于原油中轻质组分的大量挥发，分子量较大且结构复杂的石油烃组分受风化作用的影响较小。通过气相色谱分析，原油在风化过程中的质量损失主要来源于C17之前的烃组分挥发。
通过海水模拟实验柱对油污染水体环境进行了模拟。用化学消油剂处理海水表层的溢油污染物时，随着海水深度的不断增加，海水垂直方向上石油烃组分的浓度逐渐减小；用微生物修复菌剂处理海水中的溢油污染物时，在初始阶段微生物菌株会优先利用溶解态的石油烃组分进行生长代谢，之后在生长过程中产生的生物表面活性剂等代谢产物会促进油组分的溶解，水体表层石油烃浓度高于底层。
在近岸滩涂沉积物模拟实验环境中，通过室内摇瓶实验可知油砂混合物在海水的冲刷作用下经过10小时达到吸附解吸平衡，水体中石油烃组分的含量为6.40mg·L~(-1)。通过柱型模拟实验证明，在近岸滩涂溢油污染区域应加强10cm以内海砂的修复处理工作。
（3）在微生物修复室内摇瓶实验过程中（25oC，120r/min），烃降解菌N1-N4对原油的降解率为63.2%，通过对微生物降解动力学过程的考察，N系列功能菌对原油的降解较好的符合了一级动力学过程。产生物表面活性剂菌株Bbai~(-1)的加入在一定程度上加快了N1-N4对石油烃组分的降解速率，降解率为66%。
（4）微生物修复室内箱体模拟实验是对室内摇瓶实验的放大，在水体温度为20oC时，细菌总数和石油烃降解菌数量维持在10~7CFU mL~(-1)的水平。菌剂对水体表层原油的乳化效果不理想，实验池中溶解态总烃的含量为5.11mg·L~(-1)。在水体温度为25oC时室内温度为19oC，在短时间内细菌总数和石油烃降解菌数量由最初的的10~6～10~7CFU mL~(-1)上升至10~8CFU mL~(-1)，原油的物理状态从实验之初较大的块状逐渐被乳化成很小的球状或片状，水体中溶解态总烃的含量由0.129～0.370mg L~(-1)逐渐上升至32.597～34.366mg L~(-1)，乳化效果优于20oC时的乳化效果。通过GC和GC-MS检测，水体温度为20C，微生物菌剂的降油效果不明显，水体温度为25C，微生物菌剂对石油烃的降解指数可达51.1%。
（5）微生物修复野外模拟实验共进行了103天，实验开始之初对各菌株进行了大型的工业发酵，石油烃降解菌N1-N4系列的菌剂共发酵6吨，产生物表面活性剂菌株Bbai-1发酵4吨，菌剂的保质期为20天。实验池水体中细菌总数和烃类降解菌的数量都经历了先减小后增大而后再减少的过程。添加了微生物修复菌剂的各实验池水体中的细菌总数维持在10~6~10~7CFU·mL~(-1)之间，烃降解菌的数量维持在10~5CFU·mL~(-1)以上，空白池的细菌总数始终在10~4CFU·mL~(-1)左右，石油烃降解菌总数保持在10~3CFU·mL~(-1)以下。总体来看以M06池（油、N1-N4）和M01池（油、N1-N4、Bbai-1、营养盐）中的微生物长势较好。
实验过程中各实验池中细菌所产糖脂含量不断增加，以最佳修复条件下M01池代谢所产糖脂含量最高。通过GC和GC-MS检测微生物代谢所产脂肪酸主要为十六烷酸和二十二烷酸。通过分子生态学的分析可知，随着培养时间的增长，布设了微生物修复菌剂的实验池中条带的亮度和丰度都大幅度上升，变化情况同细菌总数和烃降解菌数量的测定结果相符。通过油指纹分析可知，处于最佳降解条件下的M01池中石油烃降解菌对原油的降解效果最好，平均降解指数达到57.39%，M06、M07和M08池中由于营养盐或表面活性剂的缺乏，外加实验开展期间天气复杂多变，平均降解指数分别为39.88%、30.50%、31.99%。
（6）在微生物修复滩涂现场实验中，利用沸石吸附石油烃降解菌，可以较好地耐受海水的冲刷，从而起到更长久的保菌和修复效果。T04区域细菌总数为5.51×10~7CFU·mL~(-1)，石油烃降解菌数量为1.49×10~6CFU·mL~(-1)。T03区域细菌总数为3.08×106CFU·mL~(-1)，石油烃降解菌数量为5.90×10~5CFU·mL~(-1)。
（7）在微生物修复海底现场实验中，海水表层和底层的各个位点相对于参照位点细菌总数都有不同程度的增长，底层水中的石油烃降解菌的数量要高于表层水中的烃降解菌数量，实验漂移位点的菌数要高于固定位点菌数。各位点微生物所产代谢产物的含量遵循先增加后减少的趋势，漂移位点HD06Wb和HD07Wb所测糖脂含量在第33天达到最高，分别为43.54mg·L~(-1)和42.98mg·L~(-1)，这与菌数变化趋势相对应。分子生态学分析的结果表明第14天和第35天各位点条带的丰度和亮度都比本底值有不同程度的增大，且添加的菌剂的条带在各位点中都有出现且亮度较大。
通过室内摇瓶实验、室内箱体模拟实验、野外模拟实验、滩涂现场实验以及海底现场应用等一系列完整的微生物修复实验，评价和优化了微生物修复技术在实际应用过程中的各项工艺参数，为海洋石油污染微生物修复体系的现场应用提供了技术支持。
Hydrocarbon degrading bacteria are usually screened from oil-contaminatedareas. Generally, they could performe very well in laboratory oil degradingexperiments. But the bacteria usually could not reproduce their efficienthydrocarbon-degrading ability in the field environment due to the lack of the ability toadapt to the on-site envirnment. The study abroad has reached high levels onmicrobial remediation treatment technology while the techonology is still in thelaboratory research stage at home. There are few large-scale field experiments to carryout, even less stringent standards to follow. Experiments were performed to assess theeffect of environmental factors on the viability of oil-degradation bacteria in sea water.The aim is mainly to evaluate the application feasibility and screen the most suitablehydrocarbon-degrading bacteria in a proper way. All the conclusions are as follows:
(1) Four hydrocarbon degrading bacteria were isolated from Qingdao Port:Ochrobactrum sp. N1(No.HQ231209), Brevibacillus parabrevis N2(No.HQ231210),Brevibacillus parabrevis N3(No.HQ231211), Brevibacillus parabrevis N4(No.HQ231212). N series bacteria combined with a biosurfactant producer Bbai~(-1) hashigh efficient in petroleum degrading experiment. The mixed bacteria flora couldreach into the growth stationary phase after16hours culture in the enrichmentmedium. In the oil medium, the bacteria reach into the stationary phase after20hoursculture. Cell concentration was9.12×10~9cell·mL~(-1). The optimum conditons forcrude oil degradation are determined to be pH of8.0, temperature of25oC and salinityof (NaCl%)25g·L~(-1).
(2) The initial stage of quality loss in crude oil weathering process mainlycomes from volatilization of light component. Large molecular weight and structureof the complex hydrocarbon component of oil is rearely affected by weathering.Through gas chromatography analysis of crude oil in the weathering process, lossmainly comes from the hydrocarbon component volatilization before C17.
When the oil spills are treated with chemical dispersants, the oil concentration inseawater decrease along with the increasing of water depth. When micribial agents isapplied in the treatment of oil spills, the bacteria in the initial stage only use thedissoluted oil in seawater. After the bacteria adapt to the marine environment,biosurfactant produced in the growth process could increase the dissolution of the oilcomponent. Hydrocarbon concentration in the surface of the seawater is higher thanthe one in the bottom.
In the oil migration shake flask experiment, the oil-sand mixture reach theadsorption-desorption equilibrium after10hours of washing action of the sea water.The components of petroleum hydrocarbons in water is of6.40mg·L~(-1). Columnarsimulation experiment show that the treatment of inshore oil contaminated areasshould be strengthened within10cm of sea sand.
(3) N series bacteria could degrade63.2%of the crude oil in the shake flaskexperiment. Microbial degradation of crude oil consists with first-order kinetics.Biosurfactant producer strain Bbai-1could accelerate the degradation of crude oil byN series bacteria. The degradation rate is66%.
(4) The indoor mesocosm experiment is the amplification of the shake flaskexperiment. When the temperature is20oC, TVC and the number of the HDB remainat the level of10~7CFU·mL~(-1). The dissolved hydrocarbons concentration is of5.11mg·L~(-1). When the temperature is25oC, the room temperature is19oC. TVC and thenumber of the HDB could reach up to10~8CFU mL~(-1)from10~7CFU mL~(-1). Oilconcentration in seawater is32.597to34.366mg L~(-1). The emulsification efficience isbetter than the one in the temperature of20oC. Based on the GC and GC-MS analysis,the degradation rate is51.1%in the temperature of25oC.
(5) The field simulation experiment is conducted for103days. N series bacteriaagent is fermented a total of six tons. Biosurfactant producer strain Bbai-1isfermented to be4tons. In the experimental pool, TVC and the number of HDB areexperienced a process of decreas, increas and finally decrease. TVC in eachexperimental pool maintain at the level of10~6~10~7CFU·mL~(-1). The number of HDB ismaintained at more than10~5CFU·mL~(-1). TVC and HDB in the control were 10~4CFU·mL~(-1)and10~3CFU·mL~(-1)respectively. The strains in M06(oil, the microbesare growing well in the N1-N4) and M01(oil, N1-N4, Bbai-1nutrient) grow betterthan other pools.The glycolipid content produced by the strains maintains increase ineach experimental pool. Glycolipid in M01has the hightest yield. Based on the GCand GC-MS analysis, the metabolites are mainly hexadecanoic acid and behenic acid.Molecular Ecology's analysis shows that, with the growth of the culture time,brightness and abundance of the inocula experiment pool bands increasedsignificantly. The degradation rate in M01pool was57.39%. The average degradationrates in M06, M07and M08are39.88%,30.50%,31.99%due to the lack of nutrientsor surfactant.
(6)In the bioremediation of shore site experiment, the use of zeolite adsorbinghydrocarbon degrading bacteria could tolerate the washing effect from seawater. Itcan keep the number of function bacteria in a high level. TVC and the number of theHDB in T04region are5.51×10~7CFU·mL~(-1)and1.49×10~6CFU·mL~(-1)respectively.TVC and the number of the HDB in T03region are3.08×10~6CFU·mL~(-1)and5.90×10~5CFU·mL~(-1)respectively.
(7) In the bioremediation of marine sediment experiment, TVC both in thesurface water and in the bottom increase to a certaine extent compared with thecontrol station. The number of HDB in the bottom water is higher than the one in thesurface wate. The bacteria in drifting station are more than the fixed station.
Glycolipid produed in the drifting stations HD06Wb and HD07Wb are43.54mg·L~(-1)and42.98mg·L~(-1)respectively after33days experiment. It is correspondingwith the bacteria growth trend. Molecular ecology analysis results show that theabundance and brightness on the14d and the35d are greater than the background.
Each technological parameter is optimized through the series of bioremediationexperiment from indoor shake flask experiment to marine site application. Theprogram conducted in this study is the exploration, in China, in forming a commonmethod to evaluate petroleum-utilizing abilities of bioremediation agents.
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