海洋烷烃降解菌的富集分离与多样性初步分析及其生物表面活性剂产生菌的筛选
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摘要
随着人类开发地球资源的加剧,海洋受到了严重的污染。目前普遍认为生物修复是清除海洋石油污染的有效手段。从海洋石油污染较严重的环境中进行石油烃降解微生物的分离、培养,以及对这些重要降解菌的鉴定是当前海洋环境微生物学研究的重点和热点。在众多的污染中,石油是海洋环境中的重要污染物,烷烃是石油的重要组成成分。但是其疏水性往往不利于微生物对其快速降解。化学表面活性剂虽然可以驱散油或加大其水溶性,但是由于其生物毒性,往往会阻碍微生物对油的利用。生物表面活性剂(biosurfactant)无毒、可生物降解,不会对环境造成污染,且能够增强憎水性化合物的亲水性和生物可利用性,提高微生物的数量,继而提高了烷烃的降解速度,是提高生物修复技术的有效手段。此外,生物表面活性剂在环境生物工程其它领域也有极大的应用潜力。因此,对烷烃降解菌和生物表面活性剂产生菌的研究具有较高的理论和应用价值。
本文以十六烷为唯一碳源,通过培养和非培养两种手段研究了南海沉积物中石油降解菌的多样性。通过烷烃富集培养,从2个站点不同深度的南海沉积物样品中富集筛选出48株深海细菌,分属于10个不同的属,其中27株对十六烷有降解能力。表面张力测定结果表明,4株降解菌同时具有较强的表面活性剂产生能力,2株Dietzia maris菌能使水的表面张力降至33mN/m左右,这是该种微生物产表面活性剂的首次报道。通过变性梯度凝胶电泳(DGGE)分析显示,南海沉积物富集物中的烷烃降解菌优势菌是芽孢杆菌,而且有多种。其中,Bacillus aquimaris在两个站点的7个样品的富集物中都是优势菌。此外,Sporosarcina,Halomona以及Brevibacterium属的细菌在不同样品中也表现为除Bacillus之外的优势菌。其中大多数菌已报道可以降解原油或柴油。
从南非开普敦码头的表层海水的十个样品中,以原油和柴油为唯一碳源,分离筛选89株单菌,经鉴定70株为烷烃降解菌。对十个站点中烷烃降解菌分布状况进行分析,用聚合酶链式反应(PCR)、变性梯度凝胶电泳(DGGE)和切胶测序相结合的方法对十个样品中的细菌多样性进行分析,结果显示开普敦表层海水中的烷烃降解菌优势菌是食烷菌。但是,不同样品之间存在差别。在S23站点中Sphingopyxis和Bacillus是优势菌,在S24站点中Alcanivorax和Marinobacter是优势菌,在S25站点中Marinobacter是优势菌,在S26站点中Alcanivorax,Marinobacter,Sinorhizobium和Luteibacter是优势菌,在S27站点中Alcanivorax是优势菌,在S28站点中Acinetobacter和Vibrio是优势菌,在S29站点中Acinetobacter和Erythrobacter是优势菌,在S30站点中Alcanivorax是优势菌,在S31站点中Alcanivorax和Erythrobacter是优势菌,在S32站点中Alcanivorax是优势菌。同时还有一些未培养的细菌存在。选取四株较好的烷烃降解单菌接种于十六烷作为唯一碳源的培养基中,利用气质联用(GC-MS)测定烷烃降解率。培养三天后,降解率分别为43%,37%,40%,46%。为石油污染的生物修复技术的应用提供丰富的菌种资源。
以十六烷为唯一碳源,通过测定西南印度洋中脊深海水样中降解菌单菌培养液的表面张力,筛选出27株表面活性剂产生菌。有9种与GenBank中最近缘的菌株最高相似度均低于97%,是表面活性剂产生菌的新种或新属。这些菌所产生的表面活性剂成分尚未确定,也许具有新的生物活性,具有进一步研究的价值。
With the exploitation of the earth resources, marine has been suffered from seriouspollutions. Bioremediation is now widely recognized as an effective way to remove marine oil pollution. It has long been recognized that many microorganisms can use medium or long-chain n-alkanes as sources of carbon and energy, which has stimulated many studies on the usefulness of these organisms in the bioremediation of oil spills and contaminated sites. It's very important and necessary to isolate and culture and detect petroleum-degrading microorganisms from the polluted environment. Oil is one of the most important pollutants in the marine environment, but its hydrophobicity inevitably slows the degradation by microorganisms. Although chemical surfactants can disperse oil or increase oil water-solublility, but they tends to impede the oil use by microorganisms because of their toxicity. Biosurfactants are non-toxic, biodegradable, and will not cause pollution to the environment. Meanwhile biosurfactants can enhance the bioavailability of hydrophobic and hydrophilic compounds, therefore increase the degradation rate of alkane. In addition, the biosurfactant have great potential application in the environmental protection, crude oil recovery, health care, and food-processing industries.
Deep sea sediment samples of the South China Sea were used to isolate hydrocarbon degrading bacteria. As a result, forty eight isolates were obtained from the enrichments with hexadecane as the sole carbon sources. Among them, 27 isolates were capable of degrading alkane; and 4 could produce biosurfactant significantly as determined by the surface tension measurement. Two isolates belonging to Dietzia maris lowered water surface tension to 33 mN/m. This is the first report about D. maris in biosurfactant production. The results of polymerase chain reaction (PCR) followed by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing suggested that Bacillus was the dominant member in the hexadecane enriched communities. Isolates of Bacillus aquimaris were demonstrated to be the most predominant degraders in all 7 samples at 2 sampling sites. In addition, Sporosarcina, Halomonas and Brevibacterium were also found as one of the dominant members in some samples. Therefore, species of Bacillus might play an important role in alkane degradation in te sediments of the sampling sites.
By enrichment with crude oil and diesel as the sole carbon source, the diversity of petroleum-degrading bacteria in the surface water of Cape Town was surveyed. Ten samples resulted in 10 petroleum -degrading consortia, whose structures were analyzed by the combined methods of DGGE and CFU counting. DGGE revealed quite diversity in bacterial population. We isolated 94 strains in total and they belong to 23 different genus. They belong to Proteobacteria, Acinobacteria, Flavobacteria, Firmicutes, Sphingobacteria, respectively. The results showed that the dominant strains were detected phylogenetically close to Alcanivorax, Marinobacter, Martelella, Novosphingobium, and Tthalassospira respectively. Four alkane degrading bacteria were selected to examine their potential in alkane degradation. After three days in cubation, 43%, 37%, 40% and 46% of hexadecane were degraded as quantified by GC-MS, respectively.
With two kinds of methods including measurement of surface tension and oil-displacement test, 27 biosurfactant-producing bacteria was isolated from the southwest ridge of Indian Ocean. On the basis of 16S rRNA gene sequence similarity, there are 9 strains are less than the maximum 97% similarity .Therefore, 9 novel species or genus were proposed. The surfactant composition generated by these bacteria has not yet been determined; perhaps have new biological activity, with the value of further study.
本文以十六烷为唯一碳源,通过培养和非培养两种手段研究了南海沉积物中石油降解菌的多样性。通过烷烃富集培养,从2个站点不同深度的南海沉积物样品中富集筛选出48株深海细菌,分属于10个不同的属,其中27株对十六烷有降解能力。表面张力测定结果表明,4株降解菌同时具有较强的表面活性剂产生能力,2株Dietzia maris菌能使水的表面张力降至33mN/m左右,这是该种微生物产表面活性剂的首次报道。通过变性梯度凝胶电泳(DGGE)分析显示,南海沉积物富集物中的烷烃降解菌优势菌是芽孢杆菌,而且有多种。其中,Bacillus aquimaris在两个站点的7个样品的富集物中都是优势菌。此外,Sporosarcina,Halomona以及Brevibacterium属的细菌在不同样品中也表现为除Bacillus之外的优势菌。其中大多数菌已报道可以降解原油或柴油。
从南非开普敦码头的表层海水的十个样品中,以原油和柴油为唯一碳源,分离筛选89株单菌,经鉴定70株为烷烃降解菌。对十个站点中烷烃降解菌分布状况进行分析,用聚合酶链式反应(PCR)、变性梯度凝胶电泳(DGGE)和切胶测序相结合的方法对十个样品中的细菌多样性进行分析,结果显示开普敦表层海水中的烷烃降解菌优势菌是食烷菌。但是,不同样品之间存在差别。在S23站点中Sphingopyxis和Bacillus是优势菌,在S24站点中Alcanivorax和Marinobacter是优势菌,在S25站点中Marinobacter是优势菌,在S26站点中Alcanivorax,Marinobacter,Sinorhizobium和Luteibacter是优势菌,在S27站点中Alcanivorax是优势菌,在S28站点中Acinetobacter和Vibrio是优势菌,在S29站点中Acinetobacter和Erythrobacter是优势菌,在S30站点中Alcanivorax是优势菌,在S31站点中Alcanivorax和Erythrobacter是优势菌,在S32站点中Alcanivorax是优势菌。同时还有一些未培养的细菌存在。选取四株较好的烷烃降解单菌接种于十六烷作为唯一碳源的培养基中,利用气质联用(GC-MS)测定烷烃降解率。培养三天后,降解率分别为43%,37%,40%,46%。为石油污染的生物修复技术的应用提供丰富的菌种资源。
以十六烷为唯一碳源,通过测定西南印度洋中脊深海水样中降解菌单菌培养液的表面张力,筛选出27株表面活性剂产生菌。有9种与GenBank中最近缘的菌株最高相似度均低于97%,是表面活性剂产生菌的新种或新属。这些菌所产生的表面活性剂成分尚未确定,也许具有新的生物活性,具有进一步研究的价值。
With the exploitation of the earth resources, marine has been suffered from seriouspollutions. Bioremediation is now widely recognized as an effective way to remove marine oil pollution. It has long been recognized that many microorganisms can use medium or long-chain n-alkanes as sources of carbon and energy, which has stimulated many studies on the usefulness of these organisms in the bioremediation of oil spills and contaminated sites. It's very important and necessary to isolate and culture and detect petroleum-degrading microorganisms from the polluted environment. Oil is one of the most important pollutants in the marine environment, but its hydrophobicity inevitably slows the degradation by microorganisms. Although chemical surfactants can disperse oil or increase oil water-solublility, but they tends to impede the oil use by microorganisms because of their toxicity. Biosurfactants are non-toxic, biodegradable, and will not cause pollution to the environment. Meanwhile biosurfactants can enhance the bioavailability of hydrophobic and hydrophilic compounds, therefore increase the degradation rate of alkane. In addition, the biosurfactant have great potential application in the environmental protection, crude oil recovery, health care, and food-processing industries.
Deep sea sediment samples of the South China Sea were used to isolate hydrocarbon degrading bacteria. As a result, forty eight isolates were obtained from the enrichments with hexadecane as the sole carbon sources. Among them, 27 isolates were capable of degrading alkane; and 4 could produce biosurfactant significantly as determined by the surface tension measurement. Two isolates belonging to Dietzia maris lowered water surface tension to 33 mN/m. This is the first report about D. maris in biosurfactant production. The results of polymerase chain reaction (PCR) followed by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing suggested that Bacillus was the dominant member in the hexadecane enriched communities. Isolates of Bacillus aquimaris were demonstrated to be the most predominant degraders in all 7 samples at 2 sampling sites. In addition, Sporosarcina, Halomonas and Brevibacterium were also found as one of the dominant members in some samples. Therefore, species of Bacillus might play an important role in alkane degradation in te sediments of the sampling sites.
By enrichment with crude oil and diesel as the sole carbon source, the diversity of petroleum-degrading bacteria in the surface water of Cape Town was surveyed. Ten samples resulted in 10 petroleum -degrading consortia, whose structures were analyzed by the combined methods of DGGE and CFU counting. DGGE revealed quite diversity in bacterial population. We isolated 94 strains in total and they belong to 23 different genus. They belong to Proteobacteria, Acinobacteria, Flavobacteria, Firmicutes, Sphingobacteria, respectively. The results showed that the dominant strains were detected phylogenetically close to Alcanivorax, Marinobacter, Martelella, Novosphingobium, and Tthalassospira respectively. Four alkane degrading bacteria were selected to examine their potential in alkane degradation. After three days in cubation, 43%, 37%, 40% and 46% of hexadecane were degraded as quantified by GC-MS, respectively.
With two kinds of methods including measurement of surface tension and oil-displacement test, 27 biosurfactant-producing bacteria was isolated from the southwest ridge of Indian Ocean. On the basis of 16S rRNA gene sequence similarity, there are 9 strains are less than the maximum 97% similarity .Therefore, 9 novel species or genus were proposed. The surfactant composition generated by these bacteria has not yet been determined; perhaps have new biological activity, with the value of further study.
引文
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