天津海域石油降解细菌的分离鉴定及其主要降解途径的研究
发布时间：2018-08-23 14:29
【摘要】：近年来,海洋石油污染不断加剧,海洋环境的治理已是迫在眉睫。在现有的治理海洋石油污染的措施中,生物修复法由于其安全、无二次污染等优点脱颖而出,而石油降解菌就是一类在生物修复法中扮演着重要角色的微生物。本论文在天津海域表层水样中分离出了六株烷烃降解细菌,生理生化分析及分子生物学鉴定结果显示该六株细菌均属于柠檬酸杆菌属(Citrobacter sp.)。分别命名为:Citrobacter sp.TUST-S1～Citrobacter sp.TUST-S6(GeneBank 登录号为 KM186143～KM186148)。为进一步了解所分离的六株菌株降解石油烃的能力,探究了各菌株的降解特性、乳化活性及疏水性。采用萃取技术结合GC-MS技术测定菌株的降解特性:用正己烷萃取各菌株降解前后培养基中的残油组分,除水过滤,于4 ℃保存。将C7～C30正构烷烃及异三十烷的标准品,配制成终浓度1OOmg/L后,等体积混合,并将异三十烷作为内标,取相同体积加入到个样品中,进行GC-MS分析。结果显示菌株的降解率相差很大,降解率最小的菌株为Citrobacter sp.TUST-S6,降解率只有39%;降解率最高的菌株为Citrobactersp.TUST-S5,降解率高达87%。在对菌株Citrobacter sp.TUS-T-S5的降解率具体分析时发现,该菌株能降解C11～C28的烷烃,对碳链长度大于C20的烷烃,降解率达到了 80%以上。将等体积的菌悬液与正十六烷涡旋混合后,静置分层,利用乳化活性及疏水性公式计算菌株的乳化活性及疏水性,结果显示降解率较大的菌株Citrobacte.sp.TUST-S5的乳化活性及疏水性也相对较大,分别为47.86%和38.30%,推测降解过程中,菌株产生的乳化作用及疏水作用可能会促进降解作用。鉴于Citrobacter sp.TUST-S5具有较高的降解率,以Citrobacter sp.TUST-S5为实验材料,测定了菌株的生长与降解之间的关系、降解关键酶基因(alkB)的表达模式及主要降解途径。在测定了一个生长周期内菌株降解率的变化情况后,发现菌株的降解作用与其菌浓度紧密相关。使用Q-PCR技术分析了降解关键酶基因(alkB)的表达模式,结果表明该基因的表达量,在菌株培养8小时时最高。利用GC-MS技术检测降解过程中的产物,推测可能的降解途径,结果显示参与的降解途径主要有末端氧化途径和次末端氧化途径,反应中还通过缩合反应,直接脱氢作用,末端脱水作用等生成了部分中间产物。
[Abstract]:In recent years, marine oil pollution is increasing, marine environment management is imminent. Among the existing measures to control marine oil pollution, bioremediation has the advantages of safety and no secondary pollution. Petroleum degrading bacteria are a kind of microorganism that plays an important role in bioremediation. In this paper, six alkane degrading bacteria were isolated from the surface water samples of Tianjin sea area. The results of physiological and biochemical analysis and molecular biological identification showed that the six bacteria belong to the genus (Citrobacter sp.). They are named as: Citrobacter sp.TUST-S1~Citrobacter sp.TUST-S6 (GeneBank login number is KM186143~KM186148). In order to further understand the ability of the six strains to degrade petroleum hydrocarbon, the degradation characteristics, emulsifying activity and hydrophobicity of each strain were studied. The degradation characteristics of the strains were determined by using extraction technique and GC-MS technique. The residual oil components in the culture medium before and after degradation were extracted by n-hexane and filtered by water removal and stored at 4 鈩，
本文编号：2199352
[Abstract]:In recent years, marine oil pollution is increasing, marine environment management is imminent. Among the existing measures to control marine oil pollution, bioremediation has the advantages of safety and no secondary pollution. Petroleum degrading bacteria are a kind of microorganism that plays an important role in bioremediation. In this paper, six alkane degrading bacteria were isolated from the surface water samples of Tianjin sea area. The results of physiological and biochemical analysis and molecular biological identification showed that the six bacteria belong to the genus (Citrobacter sp.). They are named as: Citrobacter sp.TUST-S1~Citrobacter sp.TUST-S6 (GeneBank login number is KM186143~KM186148). In order to further understand the ability of the six strains to degrade petroleum hydrocarbon, the degradation characteristics, emulsifying activity and hydrophobicity of each strain were studied. The degradation characteristics of the strains were determined by using extraction technique and GC-MS technique. The residual oil components in the culture medium before and after degradation were extracted by n-hexane and filtered by water removal and stored at 4 鈩，
本文编号：2199352
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