摘要
在21世纪,环保工作者们对红树林湿地以及红树林中具有海洋污染生物修复功能的微生物资源的研究已经成为一个热点。本研究的目的是探索红树林区微生物在石油污染压力下的细菌数量、代谢活性以及群落结构的变化,并从中分离筛选石油降解菌。样品采集于中国福建省九龙江口红树林湿地的3个站位,以原油和柴油为选择压力富集驯化样品中的微生物。在富集驯化过程中,测定了可培养细菌的数量、微生物的代谢活性,并用PCR-DGGE的方法分析了驯化过程中细菌群落结构的变化。结果如下:
1.经过富集驯化,从红树林沉积物中获得了大量的可培养细菌。非转接组在驯化过程的第一周到第五周,细菌的数量恒定,从第六周开始到第八周,所有实验组的细菌数量都呈缓慢的减少趋势。在转接组,15号站位可培养细菌数量最高,其次是14号站位,12号站,丰度最低的是海水的驯化样品。结果还显示,以柴油为富集压力下的样品中的平均细菌数高于原油样品,此外,所有站位样品以2216E作为富集培养基得到的细菌数均高于经R_2A培养的。
2.测定了不同站位以及富集富集驯化的微生物的电子传递系统的活性(ETSA)。在非转接组中,ETSA值从第一周到第五周基本不变,第六周到第八周开始下降。在转接组中的结果表明,15号站位的样品表现有最高的活性,其次是海水富集物),14号站位和12号站位。另外,以柴油为富集压力下的样品的ETSA平均值高于原油的。同时发现所有站位的样品在以2216E作为富集培养基得到的ETSA平均值高于R_2A。
3.利用PCR-DGGE方法分析了研究了红树林沉积物在原油和柴油污染压力下微生物群落结构的变化。DGGE的条带表明,第一周到第八周,细菌的群落结构没有明显的变化。并且,对获得的8条优势条带进行了测序,结果表明它们的分别属于Vibrio natriegens,Vibronaceae bacterium,Vibrio campbellii,Uncultured marine bactyerium,Vibrioparahaemolyticus,Pseudomonas sp,Vibrio sp和Vibrio alginolyticus。
4.从原油和柴油富集培养基中分离得到了11株菌,16S rDNA文库分析表明,绝大多数分离到得细菌其序列相对应的细菌为此前已确定的潜在的多环芳烃(PAHs)降解菌,在Gene bank比对结果表明这11株菌分别归属于Pseudomonas sp.,Flexibacteraceaebacterium,Rhodobacteraceae bacterium,Marinobacter sp.,Alteromonas sp.,MarineCFB-group bacterium,Marinobacter aquaeolei,Thalassospira sp.,Alteromonas sp.,Muricauda sp.,and Uncultured4lteromonas.其中2株的相似性低于97%,可能是潜在的新种、属。
Research about mangrove areas as well as their microbial resources for the bioremediation of oil pollution in marine environment has become a hot spot in the twenty-first century by environmentalists.This study was set out to determine the ability of oil-degrading bacteria in mangrove ecosystem to utilize diesel oil and crude oil as a carbon source in an in-situ condition in both non-transfer and transfer enrichment samples and determine their community structure.Sediment samples were collected from three stations in Fugong mangrove swamp of the Jiulong River Estuary,Fujian,China and the microorganism were enriched in MSM supplemented with crude oil and diesel oil.The metabolic activities of microorganism were analyzed and the changes in microbial community structure were evaluated using PCR- DGGE analysis of amplified 16S rDNA gene fragment during batch culture enrichment.The results obtained are as follows:
1.The mangrove sediments harbored abundant cultivatable bacteria.In the non-transfer sample,there was a constant bacteria number between the first 1st to the 5th week of enrichment and the number start declining in the 6~(th) to the 8~(th) weeks of enrichment in all the stations with little variation.In the transfer sample there was a decrease in the cultivatable bacteria number from the first week of enrichment in all the stations.
2.The Electron Transport System Activity(ETSA) of sediment samples and enrichment cultures were tested in both non-transfer and the transfer enrichment sample.In the non-transfer sample.there was a constant ETSA number between the first 1-5 weeks as shown by their OD value and the number started to decline in the 6~(th) to the 8~(th) weeks of enrichment in all the stations.In the transfer sample,the result showed a decrease in the ETSA value and station 15 exhibited high activity rate followed by the sea water enrichment,station 14 and the least was recorded at station 12.
3.The DGGE profile demonstrated that there was no remarkably shift in bacteria community from first subculture to the eighth subculture.DGGE bands were successfully sequenced and eight dominant species were identified.They were closely related to Vibrio natriegens,Vibronaceae bacterium,Vibrio campbellii, Uncultured marine bactyerium,Vibrio parahaemolyticus,Pseudomonas sp., Vibrio sp.and Vibrio alginolyticus respectively.
4.Eleven strains were isolated from the cultivatable culture dependent bacteria from both the crude oil and diesel oil enrichment culture and 16S rDNA library analysis showed that the vast majority of the corresponding sequences of cultured bacteria are microorganisms that have been previously identified as potential polycyclic aromatic hydrocarbons(PAHs) degraders,Furthermore, when the corresponding sequences were compared with the GeneBank database revealed the following populations of bacteria capable of utilizing petroleum product as a carbon source as follows;Pseudomonas sp.,Flexibacteraceae bacterium,Rhodobacteraceae bacterium,Marinobacter sp.,Alteromonas sp., Marine CFB-group bacterium,Marinobacter aquaeolei,Thalassospira sp., Alteromonas sp.,Muricauda sp.,and Uncultured Alteromonas sp.respectively.
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