【摘要】：近年來,海洋石油污染不斷加劇,海洋環(huán)境的治理已是迫在眉睫。在現(xiàn)有的治理海洋石油污染的措施中,生物修復法由于其安全、無二次污染等優(yōu)點脫穎而出,而石油降解菌就是一類在生物修復法中扮演著重要角色的微生物。本論文在天津海域表層水樣中分離出了六株烷烴降解細菌,生理生化分析及分子生物學鑒定結(jié)果顯示該六株細菌均屬于檸檬酸桿菌屬(Citrobacter sp.)。分別命名為:Citrobacter sp.TUST-S1～Citrobacter sp.TUST-S6(GeneBank 登錄號為 KM186143～KM186148)。為進一步了解所分離的六株菌株降解石油烴的能力,探究了各菌株的降解特性、乳化活性及疏水性。采用萃取技術(shù)結(jié)合GC-MS技術(shù)測定菌株的降解特性:用正己烷萃取各菌株降解前后培養(yǎng)基中的殘油組分,除水過濾,于4 ℃保存。將C7～C30正構(gòu)烷烴及異三十烷的標準品,配制成終濃度1OOmg/L后,等體積混合,并將異三十烷作為內(nèi)標,取相同體積加入到個樣品中,進行GC-MS分析。結(jié)果顯示菌株的降解率相差很大,降解率最小的菌株為Citrobacter sp.TUST-S6,降解率只有39%;降解率最高的菌株為Citrobactersp.TUST-S5,降解率高達87%。在對菌株Citrobacter sp.TUS-T-S5的降解率具體分析時發(fā)現(xiàn),該菌株能降解C11～C28的烷烴,對碳鏈長度大于C20的烷烴,降解率達到了 80%以上。將等體積的菌懸液與正十六烷渦旋混合后,靜置分層,利用乳化活性及疏水性公式計算菌株的乳化活性及疏水性,結(jié)果顯示降解率較大的菌株Citrobacte.sp.TUST-S5的乳化活性及疏水性也相對較大,分別為47.86%和38.30%,推測降解過程中,菌株產(chǎn)生的乳化作用及疏水作用可能會促進降解作用。鑒于Citrobacter sp.TUST-S5具有較高的降解率,以Citrobacter sp.TUST-S5為實驗材料,測定了菌株的生長與降解之間的關(guān)系、降解關(guān)鍵酶基因(alkB)的表達模式及主要降解途徑。在測定了一個生長周期內(nèi)菌株降解率的變化情況后,發(fā)現(xiàn)菌株的降解作用與其菌濃度緊密相關(guān)。使用Q-PCR技術(shù)分析了降解關(guān)鍵酶基因(alkB)的表達模式,結(jié)果表明該基因的表達量,在菌株培養(yǎng)8小時時最高。利用GC-MS技術(shù)檢測降解過程中的產(chǎn)物,推測可能的降解途徑,結(jié)果顯示參與的降解途徑主要有末端氧化途徑和次末端氧化途徑,反應中還通過縮合反應,直接脫氫作用,末端脫水作用等生成了部分中間產(chǎn)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，

本文編號：2199352