IOCAS-IR
南海冷泉微生物多样性分析及一株新菌的鉴定
刘施琪
Subtype硕士
Thesis Advisor孙黎
2019-05-17
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name农学硕士
Keyword深海,冷泉,南海,微生物多样性,新菌
Abstract

深海占据了约三分之二的地球总面积,是全球最大的生态系统,含有丰富的生物资源,但由于深海研究起步晚难度大,人们对这一生态系统及其中生物的了解还十分有限。冷泉系统海底碳氢化合物渗漏形成,是深海贫瘠环境中的绿洲,在世界范围内广泛存在。大多数冷泉系统富含甲烷和硫化氢,参与硫循环和甲烷循环硫氧化菌、硫酸盐还原菌和甲烷氧化古菌等微生物在该生态系统中发挥着极其重要的作用。本文针对2017科学号科学考察船南海台湾西南部冷泉区采集的水体和沉积物样品,利用非培养法和培养法对其中的微生物进行了分析。

我们通过16S rDNA高通量测序技术对来源于南海冷泉渗漏区、非渗漏区以及非冷泉区的水体和沉积物样品进行了微生物群落结构分析。通过将冷泉区样品与其他样品中的微生物进行比较分析我们发现冷泉区存在着独特的微生物群落结构:冷泉区样品中大量存在参与硫循环和甲烷循环的微生物,其含量远远高于非渗漏区和非冷泉区,且沉积物中微生物群落随深度变化表现出明显的分布特征。具体来说,冷泉渗漏区水体及表层0-8 cm沉积物样品中存在大量的硫氧化菌Sulfurovum,占总OTUoperational taxonomic unit,可操作分类单元)含量的40.78 %-77.76 %,且其在沉积物中的分布随深度增加而下降;冷泉区8-22 cm的沉积物中存在大量的硫酸盐还原菌SEEP-SRB1,冷泉区中下层10-26 cm沉积物中存在着大量的甲烷氧化菌,其中10-22 cm中主要为ANME-2a-2bANME-3,与SEEP-SRB1分布相似;22-26 cm样品中主要为ANME-1而非渗漏区水体样品中主要为α-变形菌,非冷泉区沉积物中微生物群落主要是酸微菌目。

本文利用多种培养基和多种培养方法对冷泉区沉积物样品中的微生物进行了分离培养,共分离出161株细菌,其中2株菌与已知菌株的16S rDNA相似度低于97 %阈值,推测为新种。好氧细菌共155株,分布在3个门,5个纲,13个目,23个科,26个属,60个种和亚种,其中γ-变形菌纲数目最多,共88株,包括13个属;α-变形菌纲共41株,包括5个属;芽孢杆菌纲共23株,包括5个属;噬纤维菌纲2株,包括1个属黄杆菌纲1株。厌氧细菌共2株,分别属于假单胞菌属和鞘脂单胞菌属。微好氧菌株共4株,均为Alteromonas macleodii

本文对一株来源于南海沉积物的疑似新种细菌L2做了多相分类学研究。遗传学特征分析发现L216S rDNA序列相似度最高的菌株为Muricauda marina H19-56T,相似度为96.4 %;系统发育树表明菌株L2Muricauda 属菌株聚成一个M. marina H19-56TM. antarctica Ar-22TM. taeanensis 105TM. flavescens SW-62T这四株菌形成一个进化支。表型特征分析表明,菌株L2与参考菌株的脂肪酸成分、极性脂成分及DNA G+C mol%均相似,但在生长温度范围、部分大分子水解能力、酶活性和抗生素敏感性等方面存在较大差异。以上结果表明,菌株L2Muricauda属的一个新种,将菌株L2命名为Muricauda iocasae sp. nov,模式菌株为L2T (=CCTCC AB 2017193T=KCTC 62196T)

通过本研究,我们进一步了解了南海冷泉区微生物群落多样性及结构特征,发现了冷泉区功能微生物种群,这些发现有利于我们更好地理解微生物在冷泉生态系统物质能量循环过程中发挥的作用。同时,分离的细菌及发现的新种对海洋微生物资源的开发与利用具有重要意义。

Other Abstract

Deep sea is the largest habitat with rich biological resource, accounting for about two-thirds of the earth’s surface. However, due to the difficulty of research, knowledge about this ecological environment and its biological feature is very limited. The cold seep system, a fluid formed by the leakage of hydrocarbon from the seabed, is an "oasis" in the barren environment of the deep sea, widely existing in the world. Most of cold seeps are characterized by high concentrations of methane and hydrogen sulfide. Microorganisms, such as sulfur-oxidizing bacteria, sulfate-reducing bacteria and methane-oxidizing archaea, play extremely important roles in sulfur and methane cycling process. In this study, both culture-independent and culture-dependent methods were used to analyze the microbial diversity in the water and sediment samples collected in 2017 from the cold seep located at southwest Taiwan of the South China Sea.

Through 16S rDNA high-throughput sequencing technology, the microbial structure in the water and sediment samples from the cold deep, non-leakage area and non-cold seep area of the South China Sea were analyzed. The results showed that there was a unique microbial structure in the cold seep area. In the cold seep area, the relative abundance of microorganisms involved in sulfur and methane cycle were much higher than that in non-leakage zone and non-cold seep zone and varied with depth. To be specific, the water samples and sediment samples of the cold seep with depth of 0-8 cm were dominated by sulfur-oxidizing bacteria Sulfurovum, accounting for 40.78 %-77.76 % of the total OTUs (operational taxonomic unit) of the sample, and decreased with the depth. The sediment samples of the cold seep with depth of 8-22 cm contained plenty of sulfate reducing bacteria SEEP-SRB1. The sediment samples of the cold seep with the depth of 10-26 cm contained abundant methane-oxidizing archaea, of which, ANME-2a-2b and ANME-3 were detected in the depth of 10-22 cm, which was similar to the distribution of SEEP-SRB1, while ANME-1 appeared in the depth of 22-26 cm. The microbes in the water samples of non-leakage zone were mainly α-Proteobacteria, and the microbes in non-cold seep samples were mainly Acidimicrobiia.

Several cultural media and methods were used to cultivate microorganisms isolated from the cold seep sediment sample of the South China Sea. A total of 161 strains were isolated and identified. According to 16S rDNA sequences analysis, 2 strains were speculated to be new species. The 155 aerobic isolated strains belonged to 3 phyla, 5 classes, 13 orders, 23 families, 26 genera and 60 (sub)species. Gammaproteobacteria occupied the largest proportion, containing 88 strains belonging to 13 genera, followed in abundance by Alphaproteobacteria (41 stains, 5 genera), Bacilli (23 stains, 5 genera), Cytophagia (2 strains, 1genus) and Flavobacteria (1 strains, 1 genus). The 2 anaerobic strains belonged to Pseudomonas and Sphingomonas. All 4 microaerophilic strains belonged to Alteromonas.

In this study, we also characterized strain L2T, a novel species of the genus Muricauda isolated from the deep sea sediments. Analysis of 16S rDNA sequence (1423bp) indicated that strain L2T exhibited the highest sequence identity to M. marina H19-56T (96.4 %). Phylogenetic analysis showed that strain L2T fell within the clade comprising various Muricauda species. Within the clade, strain L2T formed a group with four other Muricauda species (M. marina H19-56T, M. antarctica Ar-22T, M. taeanensis 105T and M. flavescens SW-62T). The chemotaxonomic profiles of strain L2T were generally similar to those of the genus Muricauda. However, phenotypic characteristics, including the temperature range for growth, hydrolysis of some macromolecules, some enzyme activities and antibiotic susceptibilities, distinguished strain L2T from other related Muricauda species. Therefore, strain L2T represents a novel species of the genus Muricauda, for which the name Muricauda iocasae sp. nov. is proposed with the type species L2T (=CCTCC AB 2017193T =KCTC 62196T).

Through this study, we have learned more about the diversity of microbial communities in the cold seep ecosystem. The unique microbial population of the leak zone was explored. The results promote the understanding the function of microbes in the process of material and energy cycle in the cold seep. Meanwhile, novel species and other bacteria isolated and identified in this study will be useful to the development and utilization of marine microbial resources.

Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/156845
Collection中国科学院海洋研究所
实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
刘施琪. 南海冷泉微生物多样性分析及一株新菌的鉴定[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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