摘要
多环芳烃(PAHs)是一类广泛分布于海洋环境中的典型的持久性有机污染物(POPs),通过食物链的传递会对生态环境和人体健康造成极大危害。了解PAHs在环境中的来源、分布、归宿及去除问题,已成为当今环境科学研究的前沿课题。生物降解是环境(土壤,沉积物,水体)中PAHs去除的最主要途径。研究其遗传控制方式,探索代谢途径,是为了更好地将降解菌应用于有效而可控的生物修复之中。
本论文将采集自厦门博坦油码头的海水样品,在持续性的高浓度、高分子量PAHs选择压力下进行富集驯化,获得高分子量PAHs的降解混合菌系,并以此讨论高分子量多环芳烃(HMW-PAHs)降解菌的筛选策略;同时对筛选获得的2株能高效降解PAHs的细菌从生理生化角度、基因组学、蛋白质组学方面进行较为系统的研究,探讨其降解特性,解析相关功能基因及功能酶,并在这些基础上推测其代谢途径。获得的主要结果如下:
1.用高浓度(1000 mg/L),混合PAHs(菲,芘,荧蒽,苯并(a)芘)作为选择压力,从采自石油污染地区样品中驯化获得混合降解菌系,从中分离得到3株可培养单菌,变性梯度凝胶电泳(PCR—DGGE)跟踪分析驯化过程中的细菌群落结构变化及优势菌形成过程。采用高效液相色谱(HPLC)测定混合菌系BL和3株单菌BL01,BL02,BL03对高分子量、难降解的PAH——苯并(a)芘的降解能力,结合DGGE结果分析菌系中各单菌在降解过程中的作用。结果显示BL02,BL03不具有降解BaP的能力,BL01 14 d后降解了20.98%,三菌混合培养物降解了22.61%;而混合菌系BL 14天后降解了44.07%的BaP,这在同类报道中处于较高水平,显示了BL对BaP有较好的降解能力。该筛选策略有助于关注那些未可培养细菌在HMW-PAHs降解中的作用,因此采用高浓度、高分子量PAHs的选择压力筛选HMW-PAHs的降解菌是一种快速而行之有效的筛选策略。
2.对采自厦门海域的水样和沉积物样品,经过一定时间的PAHs富集培养之后,采用改良的平板升华法成功地筛选到两株菲高效降解菌,降解能力测定显示,它们都能以菲和荧蒽作为唯一碳源生长,72 h后均能完全降解起始浓度为100mg/L的菲。16S rDNA鉴定结果显示这两株分别为鞘氨醇单胞菌Sphingomonas sp.B2-7和分支杆菌Mycobacterium sp.S8。
3.通过降解菌对芳香烃化合物和PAHs降解过程中常见中间产物的利用情况,菌株B2-7能利用水杨酸,2—萘酚,邻苯二酚,初步推测它以水杨酸途径代谢菲,菌株S8不能降解萘,能利用原儿茶酸,但是不能利用邻苯二甲酸,很可能是以邻苯二甲酸以外的途径代谢菲。降解菌降解荧蒽的优化条件实验结果显示,非离子表面活性剂Tween-80和营养物葡萄糖能促进菌株B2-7和S8对荧蒽的降解。
4.芳环羟基化双加氧酶和芳环断裂双加氧酶是PAHs降解过程中开环的两种关键酶,其中环羟基化双加氧酶(RHDs)控制苯环加氧,是微生物降解反应的限速步骤,邻苯二酚双加氧酶是催化苯环开裂的重要酶。我们通过引物设计的方法,从降解菌Sphingomonas sp.B2-7中扩增得到全长的邻苯二酚-2,3-双加氧酶基因(C23O)和环羟基化双加氧酶大亚基(phnA),从分子生物学角度证明了PAHs降解过程中的重要酶系邻苯二酚-2,3-双加氧酶(C23O)和环羟化双加氧酶基因α亚基(phnA)在菌株B2-7中的存在。对phnA基因构建了重组表达载体,成功在表达宿主大肠杆菌E.coli BL21(DE3)中进行表达,基因在宿主中以溶解蛋白形式存在。可以进一步推断菌株B2-7降解菲的基本途径是RHDs氧化PAHs形成二氢二醇PAHs化合物,再在C23O的作用下经由水杨酸途径间位裂解(meta-cleavage)苯环,生成2-羟基粘康酸半醛(2-HMS)。
5.采用1-DE(一向凝胶电泳)和2-DE(双向凝胶电泳)结合LC-MS/MS(串联质谱),研究菌株B2-7在菲诱导与无菲诱导(对照)状况下的蛋白质表达,对差异蛋白的生物质谱结果进行分析,得到多个代谢途径中的关键酶(加氧酶、脱氢酶、醛缩酶等),结合菌株B2-7的降解特性与降解基因的研究结果,最终推导出菌株B2-7降解菲较为完整的代谢途径。
The presence of polycyclic aromatic hydrocarbons (PAHs) in the environment is a considerable public health hazard because of their intrinsic chemical stability, high recalcitrance to different types of degradation and high toxicity to living organisms. The principle processes for their successful removal are currently believed to be microbial transformation and degradation.
This research aimed at finding bacteria capable of breaking down the PAHs, identifying the functional genes and enzymes involved in the biodegradation, and elucidating PAHs biodegradative pathway.The main results were as follows:
1. A microbial consortium was obtained from the enrichment culture of sea water samples collected from Botan oil port in Xiamen, China, using the persistent high concentration of high molecular weight polycyclic aromatic hydrocarbons (HMW PAHs) enrichment strategy. Denaturing Gradient Gel Electrophoresis (DGGE) was adopted to investigate the bacterial composition and community dynamic changes based on 16S rRNA genes PCR amplification during enrichment batch culture. Using the spray-plate method, three bacteria namely BL01, BL02 and BL03, which corresponded to the dominant bands in the DGGE profiles, were isolated from the consortium. Sequences analysis showed BL01, BL02 and BL03 were phylogenetically closer to Ochrobactrum sp., Stenotrophomonas maltophilia and Pseudomonas fluorescens respectively. The degradation of Benzo(a)Pyrene (BaP), a model HWM-PAH compound by individual isolate, mixture of three isolates and the microbial consortium(BL) isolated from sea water were examined. Results show that the degradation capability of bacteria represented the order of consortium(BL), mixture of three isolates, individual isolate for high to low, the consortium BL showed the highest PAH degradation capability for degrading HMW-PAH compounds, 44.07% of benzo(a)pyrene was degraded by consortium BL after 14 days incubation. Our results pointed out that the high selective pressure strategy is feasible and effective to enrich the HMW PAH-degraders from the original sea water sample.
2. Using modified sublimation method, an effective and more convenient method, two phenanthrene-degrading bacteria, identified as Sphingomonas sp. B2-7 and Mycobacterium sp. S8, were isolated from a mixed culture that had been enriched under the selective pressure of PAHs. They can utilize a wide range of PAHs made of 2-4 aromatic rings as the sole carbon source and degrade these PAH compounds. Both of these two strains can degrade phenanthrene to undetectable levels in 72 h. Further experiments were carried out to optimize the degradation of HMW-PAHs and results showed that non-ionic surfactant Tween-80 and glucose can significantly increase the biodegradation percentages.
3. Strain B2-7 was used to study the enzymes involved in PAHs degradation using genomic and proteomic methods. The genes encoding two key enzymes were cloned and one of them was overexpressed in Escherichia coli BL21 (DE3). One is ring-hydroxylating dioxygenase a subunit (phnA) , the enzyme that catalyzes the initial step in PAHs degradation, and the other is catechol-2,3-dioxygenase (C23O), catalyzes the extradiol ring-cleavage of catechol. These results indicated the main catabolic pathway of strain B2-7 degrades PAHs.
4. Phenanthrene, a tricyclic PAH, was selected as the model PAH compound to determine the degradation pathway. Two-dimensional (2D) gel electrophoresis of phenanthrene-induced proteins from cultures of strain B2-7 was used to detect proteins that increased after phenanthrene exposure. Comparison of proteins from phenanthrene-induced and uninduced cultures on 2D gels indicated that at least ten major proteins were expressed. Proteins newly induced by phenanthrene were then analyzed from the gels by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Most of them shared a high degree of similarity with the existing database and were responsible for phenanthrene or naphthalene degradation, such as hydroxylating dioxygenase, dihydrodiol dehydrogenase, oxovalerate aldolase and isocitrate dehydrogenase. A complete phenanthrene degradation pathway for Sphingomonas sp. B2-7 based on these results was proposed.
引文
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