双齿围沙蚕消化道降油细菌的分离鉴定及菌群多样性分析
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摘要
双齿围沙蚕(Pernereis aibuhitensis)隶属于环节动物门、多毛纲，是重要的海洋沉积食性无脊椎动物，其迁徙距离短，在我国河口沉积物和沿海滩涂有着广泛的地理分布。双齿围沙蚕能够蓄积大量环境中的有机污染物并通过自身的生物转化作用代谢和分解污染物，沙蚕也可以改善养殖水体底质，减轻和修复环境污染，是海洋沉积环境早期污染生态风险评价的指示生物和生态毒理学研究中重要的模式生物。本研究通过微生物分离培养的方法定向筛选出双齿围沙蚕消化道共栖石油降解菌，并通过DGGE的方法分析了所分离石油降解菌在双齿围沙蚕消化道的分布情况及其肠道共栖菌群多样性。
实验将采自辽宁盘锦地区的双齿围沙蚕置于实验室无菌海水暂养24h后，将提取的沙蚕消化道样品匀浆后稀释用平板法涂布于2216E固体平板培养基，25℃培养24-48小时，根据不同菌落形态挑取单菌落进行纯化，共分离获得7株不同菌落形态的菌株，其中3株菌可在以柴油为唯一碳源的平板培养基中生长，编号分别为SC11-1、SC11-2、SC11-3,同样条件下SC11-3菌株生长状态最好，作为实验菌株。通过形态学、生理生化特性以及16SrDNA序列比对的方法对该菌株进行鉴定并测定其生长特性；同时测定添加不同葡萄糖浓度该菌株对柴油的降解性能；为进一步揭示以双齿围沙蚕为代表的多毛类消化道微生物菌群结构和分布相关生物学特征，阐明海洋底栖动物对持久性有机污染物的生物利用性和耐受性机理，实验分别从5条双齿围沙蚕消化道提取微生物基因组总DNA，通过细菌16SrDNA通用引物341F/534R进行了细菌V3高变异区的PCR扩增，再将PCR产物（约230bp）进行变性梯度凝胶电泳（DGGE），从而获得双齿围沙蚕消化道共栖细菌群落特征的DGGE条带；分别对DGGE图谱中公共条带的序列进行测序比对，分析其消化道的细菌多样性特征。结果显示：菌株SC11-3经过在25℃振荡培养（150rpm/min）15天后，对柴油的原始降解率约为40%；SC11-3菌株为革兰氏阴性菌、杆状，生理生化特征与游海假交替单胞菌(Peudoalteromonas haloplanktis)相符，16SrDNA同源性为100%；SC11-3菌株最适生长温度和pH分别为25℃和pH8，适宜NaCl浓度在1-3%之间；添加不同浓度的葡萄糖后，其降解效率有显著提高，葡萄糖添加量达到4g/L时，可降解柴油50mg以上，继续增加葡萄糖浓度，对降解率有抑制作用；通过对指纹图谱的半定量分析发现，双齿围沙蚕消化道共栖细菌菌群多样性丰富，优势条带明显，不同双齿围沙蚕个体间既存在着共同的细菌种属，也有各自特异的种属，DGGE图谱分析结果显示5条沙蚕肠道样品扩增条带数分别为22、21、18、18、19，其中样品间存在共同的优势条带，但优势条带含量存在个体间的差异；公共条带测序有下列十种细菌：假交替单胞菌属（Pseudoalteromonas sp.）未培养假交替单胞菌（Uncultured Pseudoalteromonas sp.）,伊丽莎白菌属（Elizabethkingiasp.）,产丙酸菌属（Uncultured Propionigenium sp.）,未培养细菌（Unculturedbacterium）,杆菌属（Bacterium）,未培养假交替单胞（Uncultured Pseudoalteromonassp.）,交替单胞菌科（Alteromonadaceae bacterium）,未培养γ-变形菌纲（Unculturedγ-proteobacterium）,假交替单胞菌属（Pseudoalteromonas sp.），其中产丙酸菌属（Propionigenium sp.）和假交替单胞菌属（Pseudoalteromonas sp.）分别为5条双齿围沙蚕肠道样品中的优势菌。
Polychaetes are important model organisms of marine invertebrates in estuarine sediments witha wide geographical distribution. Considering their short-distance migration characteristic,polychaetes are known to accumulate significant amounts of organic matter from theenvironment and steady-state body burdens are a function of biotransformation and eliminationprocesses. In the present study, methods of microbial cultivation were used to isolate thepetroleum-degrading bacteria in the digestive tract of Perinereis aibuhitensis (Polychaete),furthermore, DGGE was also played to analyse the distribution of isolated bacteria and theintestinal microbial diversity.
In the present study, the Perinereis aibuhitensis were collected from the shoreline of Panjin(Liaoning Province, China) and transformed to the laboratory. After being starved for24hours,the gut samples of5healthy worms were removed and the diluted homogenate was daubed onsolid2216E agar plate media. Then incubated at25℃for24-48h,7strains were eventuallyselected and purified according to different colony morphology and3of them can grow on themineral medium supplemented with diesel oil as a sole source of carbon, number SC11-1、SC11-2、SC11-3. and SC11-3strain was selected as the test strain for its relatively good growth.After determining degradation efficiency quantitatively by UV spectrophotometry, strain SC11-3was selected as the test strain for its relatively good growth and degradation ability. Detailedanalysis of morphological, biochemical and16S rDNA sequence were used to identify the isolateand its growth characteristics were determined, meanwhile the oil-degrading efficiency was alsostudied associated with different concentrations of glucose. Moreover, in order to further revealthe intestinal microflora structure-distribution related biological characteristics of Pernereisaibuhitensis presented as polychaetes, stating the bioavailability and tolerability mechanisms ofpersistent organic pollutants of marine sedimentary inverabrates, the total genomic DNA wasisolated from gut samples of5healthy Perinereis aibuhitensis, respectively. Then the V3regionof the16S rRNA genes (approximately230bp) was amplified with specific primers set ofGC-341f/534r. The DGGE band profiles of immobilized bacteria in the digestive tract ofPerinereis aibuhitensis were finally generated. The results indicate: strain SC11-3wasGram-negative, rod shaped and16S rRNA sequencing showed that strain SC11-3was closelyrelated to Peudoalteromonas haloplanktis, and the homology was100%. The experimentalresults of growing characteristics showed the optimum growth temperature and pH of strainSC11-3was25℃and8, respectively; the adaptive NaCl concentration was in the range of1-3%.After being oscillation incubated (150rpm/min) at25℃for15days, about40%diesel oil wasinitially consumed by SC11-3strain. The degrading efficiency of strain SC11-3was significantlyenhanced when adding glucose to a specific concentration of0.4g/L, and the degradation of diesel achieved more than50mg. But as more glucose was added, the degradation efficiency
dropped. The semi-quantitative analysis of DGGE map showed that22,21,18,18,19bandswere generated in5gut samples, respectively and the common predominant band was found, butthe relative content of each predominant band was different. The sequencing and blast results ofthe DGGE common bands are listed as follows: Pseudoalteromonas sp., UnculturedPseudoalteromonas sp., Elizabethkingia sp., Uncultured Propionigenium sp., Unculturedbacterium, Bacterium, Uncultured Pseudoalteromonas sp., Alteromonadaceae bacterium,Uncultured γ-proteobacterium, Pseudoalteromonas sp. and the results indicate thatPropionigenium sp. is the predominant intestinal microflora in the5samples, respectively andPseudoalteromonas sp. is widely distributed in the digestive tract of Perinereis aibuhitensis.
实验将采自辽宁盘锦地区的双齿围沙蚕置于实验室无菌海水暂养24h后，将提取的沙蚕消化道样品匀浆后稀释用平板法涂布于2216E固体平板培养基，25℃培养24-48小时，根据不同菌落形态挑取单菌落进行纯化，共分离获得7株不同菌落形态的菌株，其中3株菌可在以柴油为唯一碳源的平板培养基中生长，编号分别为SC11-1、SC11-2、SC11-3,同样条件下SC11-3菌株生长状态最好，作为实验菌株。通过形态学、生理生化特性以及16SrDNA序列比对的方法对该菌株进行鉴定并测定其生长特性；同时测定添加不同葡萄糖浓度该菌株对柴油的降解性能；为进一步揭示以双齿围沙蚕为代表的多毛类消化道微生物菌群结构和分布相关生物学特征，阐明海洋底栖动物对持久性有机污染物的生物利用性和耐受性机理，实验分别从5条双齿围沙蚕消化道提取微生物基因组总DNA，通过细菌16SrDNA通用引物341F/534R进行了细菌V3高变异区的PCR扩增，再将PCR产物（约230bp）进行变性梯度凝胶电泳（DGGE），从而获得双齿围沙蚕消化道共栖细菌群落特征的DGGE条带；分别对DGGE图谱中公共条带的序列进行测序比对，分析其消化道的细菌多样性特征。结果显示：菌株SC11-3经过在25℃振荡培养（150rpm/min）15天后，对柴油的原始降解率约为40%；SC11-3菌株为革兰氏阴性菌、杆状，生理生化特征与游海假交替单胞菌(Peudoalteromonas haloplanktis)相符，16SrDNA同源性为100%；SC11-3菌株最适生长温度和pH分别为25℃和pH8，适宜NaCl浓度在1-3%之间；添加不同浓度的葡萄糖后，其降解效率有显著提高，葡萄糖添加量达到4g/L时，可降解柴油50mg以上，继续增加葡萄糖浓度，对降解率有抑制作用；通过对指纹图谱的半定量分析发现，双齿围沙蚕消化道共栖细菌菌群多样性丰富，优势条带明显，不同双齿围沙蚕个体间既存在着共同的细菌种属，也有各自特异的种属，DGGE图谱分析结果显示5条沙蚕肠道样品扩增条带数分别为22、21、18、18、19，其中样品间存在共同的优势条带，但优势条带含量存在个体间的差异；公共条带测序有下列十种细菌：假交替单胞菌属（Pseudoalteromonas sp.）未培养假交替单胞菌（Uncultured Pseudoalteromonas sp.）,伊丽莎白菌属（Elizabethkingiasp.）,产丙酸菌属（Uncultured Propionigenium sp.）,未培养细菌（Unculturedbacterium）,杆菌属（Bacterium）,未培养假交替单胞（Uncultured Pseudoalteromonassp.）,交替单胞菌科（Alteromonadaceae bacterium）,未培养γ-变形菌纲（Unculturedγ-proteobacterium）,假交替单胞菌属（Pseudoalteromonas sp.），其中产丙酸菌属（Propionigenium sp.）和假交替单胞菌属（Pseudoalteromonas sp.）分别为5条双齿围沙蚕肠道样品中的优势菌。
Polychaetes are important model organisms of marine invertebrates in estuarine sediments witha wide geographical distribution. Considering their short-distance migration characteristic,polychaetes are known to accumulate significant amounts of organic matter from theenvironment and steady-state body burdens are a function of biotransformation and eliminationprocesses. In the present study, methods of microbial cultivation were used to isolate thepetroleum-degrading bacteria in the digestive tract of Perinereis aibuhitensis (Polychaete),furthermore, DGGE was also played to analyse the distribution of isolated bacteria and theintestinal microbial diversity.
In the present study, the Perinereis aibuhitensis were collected from the shoreline of Panjin(Liaoning Province, China) and transformed to the laboratory. After being starved for24hours,the gut samples of5healthy worms were removed and the diluted homogenate was daubed onsolid2216E agar plate media. Then incubated at25℃for24-48h,7strains were eventuallyselected and purified according to different colony morphology and3of them can grow on themineral medium supplemented with diesel oil as a sole source of carbon, number SC11-1、SC11-2、SC11-3. and SC11-3strain was selected as the test strain for its relatively good growth.After determining degradation efficiency quantitatively by UV spectrophotometry, strain SC11-3was selected as the test strain for its relatively good growth and degradation ability. Detailedanalysis of morphological, biochemical and16S rDNA sequence were used to identify the isolateand its growth characteristics were determined, meanwhile the oil-degrading efficiency was alsostudied associated with different concentrations of glucose. Moreover, in order to further revealthe intestinal microflora structure-distribution related biological characteristics of Pernereisaibuhitensis presented as polychaetes, stating the bioavailability and tolerability mechanisms ofpersistent organic pollutants of marine sedimentary inverabrates, the total genomic DNA wasisolated from gut samples of5healthy Perinereis aibuhitensis, respectively. Then the V3regionof the16S rRNA genes (approximately230bp) was amplified with specific primers set ofGC-341f/534r. The DGGE band profiles of immobilized bacteria in the digestive tract ofPerinereis aibuhitensis were finally generated. The results indicate: strain SC11-3wasGram-negative, rod shaped and16S rRNA sequencing showed that strain SC11-3was closelyrelated to Peudoalteromonas haloplanktis, and the homology was100%. The experimentalresults of growing characteristics showed the optimum growth temperature and pH of strainSC11-3was25℃and8, respectively; the adaptive NaCl concentration was in the range of1-3%.After being oscillation incubated (150rpm/min) at25℃for15days, about40%diesel oil wasinitially consumed by SC11-3strain. The degrading efficiency of strain SC11-3was significantlyenhanced when adding glucose to a specific concentration of0.4g/L, and the degradation of diesel achieved more than50mg. But as more glucose was added, the degradation efficiency
dropped. The semi-quantitative analysis of DGGE map showed that22,21,18,18,19bandswere generated in5gut samples, respectively and the common predominant band was found, butthe relative content of each predominant band was different. The sequencing and blast results ofthe DGGE common bands are listed as follows: Pseudoalteromonas sp., UnculturedPseudoalteromonas sp., Elizabethkingia sp., Uncultured Propionigenium sp., Unculturedbacterium, Bacterium, Uncultured Pseudoalteromonas sp., Alteromonadaceae bacterium,Uncultured γ-proteobacterium, Pseudoalteromonas sp. and the results indicate thatPropionigenium sp. is the predominant intestinal microflora in the5samples, respectively andPseudoalteromonas sp. is widely distributed in the digestive tract of Perinereis aibuhitensis.
引文
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