南海北部九龙甲烷礁邻区沉积物层中垂向细菌群落结构特征研究

本研究应用微生物16S rRNA-DGGE和T-RFLP技术,结合环境参数,对我国天然气水合物潜在区南海九龙甲烷礁附近973-4柱状样沉积物中3个层位12个不同深度(表层20 cm至382 cm,中层552 cm至796 cm,深层862 cm至1 196 cm)细菌群落结构及其分布进行了对比研究.其中T-RFLP实验表明,细菌丰度、香农指数和均匀度变化趋势相同,由深层到716 cm处先降后升,中层716 cm深度范围处微生物群落丰度、均匀度、香农指数相对较高,716 cm至表层先降后升.DGGE图谱和T-RFLP色谱峰聚类分析表明:表层20 cm至192 cm相似性较高,表层236 cm至382 cm与深层1 082 cm、1 196 cm群落结构相似性较高,但中层沉积物中微生物群落结构与表层及深层均有较大差异.环境参数表明中层甲烷含量较高,推测甲烷是影响微生物群落结构差异的主要因素之一.T-RFLP色谱峰与微生物数据库比对及DGGE条带测序也表明了:本区变形杆菌(Proteobacteria)为优势菌群,其中α-、γ-、δ-变形杆菌(Proteobacteria)为主要的细菌亚群,其他细菌包括放线菌(Actinobacteria)、厚壁菌门(Firmicutes)、绿弯菌门(Chloroflexi)为次优势菌群.甲烷含量较高的中层,甲烷氧化菌(Methanotrophs),硫还原菌(Sulfate-reducing bacteria)等与甲烷密切相关的细菌均有被检测到,表明该区域存在与天然气水合物的分解释放相关的微生物群落.

Vertical Microbial Community Structure Characteristics of Sediment in Gas Hydrate Potential Area of Northern South China Sea Jiulong Methane Reef

Although the marine sediments bacteria play an important role in marine environment, a comprehensive view of community characteristics is still lacking, to understand the characteristics of microbial community structure in gas hydrate potential region and to evaluate how bacterial community structure response to gas hydrate, we used PCR-based technology: terminal restriction fragment length polymorphism(T-RFLP) and denaturing gradient gel electrophoresis (DGGE), combine environment parameters such as sediment grain size, total organic carbon(TOC), deposition rate and sediment age to research the diversity of bacterial communities and structure in sediments from the adjacent region of Jiulong Methane Reef 973-4 in the northern South China Sea, and we divided the 973-4 core into three parts in the base of environmental parameters: surface layer, contain 20 cm, 192 cm, 236 cm, 326 cm, 382 cm; middle layer, contain 552 cm, 592 cm, 716 cm, 796 cm; and deep layer, contain 862 cm, 1 082 cm, 1 196 cm.The environment parameters show that the total organic carbon content decrease from surface to deep, and the content was between 0.20% to 1.83%, mostly greater than 0.5%, it can provide source for the formation of gas hydrate; the average sediment grain is between 6 μm to 8 μm; the age of the bottom of 973-4 core is 43 431 a, reveal to oxygen isotopic Ⅲ period, and the average deposition rate of this core was 34.6 cm·ka-1, meet the deposition rate of natural gas hydrate formation conditions.The result of T-RFLP show that there were much higher values of richness, Shannon-wiener index and evenness index in surface layer sediments and deep layer than the middle layer, but 716 cm was higher than other depths sediments in middle layer by the analysis of terminal restriction fragment(T-RFs). The Shannon-wiener index in some depth was lower than 2.0, show the low diversity of microbial. The three parameters have the same trend. Three parameters increased initially then reduced from deep layer to 716 cm, and the trend from 716 cm to surface layer was decrease and then increase. Though clustering result on the terminal restriction fragments(T-RFs) areas and denaturing gradient gel electrophoresis patterns shows that: the surface sediments 20 cm to 192 cm have higher similarity, the value is 52%, 236 cm were similar to 1 196, the similarity is 76%, 382 cm to 1 082 cm similarity is 38%, besides have similarity with 326 cm, 552 cm, 796 cm; the different depths in middle layer have similarity, simultaneously 716 cm and 862 cm were higher similar, this is connection with the methane level in middle layer, and methane can affect the community composition of microbial. Middle layer located in oxygen isotopicⅡ period, the template is cool, it led to the sea levels drop and made the change of the methane, and the change of methane concentration lead to great difference of microbial community structure in middle with surface and deep layers. The deep sediments microbial community has higher similarity with the surface layer because of the methane concentration was lower than middle layer and the similar total organic carbon content. Through clustering analysis and sediment environment parameters indicated that the vertical distribution of bacterial was coincided with the sediment total organic carbon content, grain size and methane concentration. And the difference of microbial community structure related to the geologic conditions for example depth, the change of sediment properties, methane concentration.Through compare T-RFLP chromatogram to Mica3 database and sequencing the DGGE bands to analysis the diversity of the sediments and the result showed that:Proteobacteria were the dominant phylum, and α-,γ-,δ-Proteobacteria were the main class within Proteobacteria, other bacteria include Actionmycetes, Firmicutes and Chloroflexi. The dominate bacteria in surface layer is δ-Proteobacteria, most of microbial were related to the hydrothermal vent of deep sea sediments, and δ-Proteobacteria have related with sulfur cycle, indicated that sulfide metabolism especially the sulfate reduction was important process; in middle layer α-Proteobacteria, δ-Proteobacteria were the dominant bacteria and almost related to the deep-sea methane seep and pacific deep-sea sediment, suggest that sulfate reduction and methane oxidation were the main process in middle layer; α-Proteobacteria was dominated in deep layer, we conclude that methane oxidation was the main process, indicating that our deep-sea sampling might be influence by the gas hydrate. In addition, methane oxidizing bacteria such as Methanotrophs and Sulfate-reducing bacteria occur in middle layer where has higher methane concentration areas, and the bacterial were related to the bacterial retrieved from sulfide chimneys, hydrothermal vent environment and areas where has confirm existing gas hydrate, indicated that the microbial community structure in core 973-4 have relation with gas hydrate decomposition and release, suggest that this zone maybe exist gas hydrate. Our data suggest that other bacteria are also involved in methane oxidation in these environments, and combine with the environment parameters we conclude that this zone exist gas hydrate, and conclusion the sulfate reduction methane oxidation transform zone located in the depths of 500 cm to 900 cm.