敦煌莫高窟第98窟壁画表面菌斑的群落结构分析

Community structures of bacterial colonies on wall paintings in Cave 98 at Mogao Grottoes, Dunhuang

摘要:

【目的】确定第98窟壁画表面白色污染物内微生物微观特征, 分析其群落组成、结构特点及诱发壁画病害微生物产生的因素, 为石窟寺保护和旅游管理提供建议。【方法】利用无菌手术刀收集壁画表面白色污染物样品; 利用扫描电子显微镜(SEM)分析样品中微生物体微观形貌; 通过提取样品总DNA、扩增细菌16S rDNA、构建克隆文库、测序和系统发生关系分析等技术研究壁画微生物群落组成与结构特点。【结果】壁画白色污染物中存在大量具有微生物特征的结构体, 形态多呈短杆状和卵圆形, 大小在 (3.0?5.5)?μm×(1.5?2.5) μm之间。共得到克隆文库序列111条, 主要为变形菌门γ-变形菌亚门肠杆菌科(Enterobacteriaceae)与假单孢菌科(Pseudomonadaceae)成员。群落组成和结构分析表明所得序列主要隶属于肠杆菌属(Enterobacter)、埃希菌属(Escherichia)、固氮菌属(Azotobacter)、沙雷氏菌属(Serratia)和克雷伯菌属(Klebsiella); 埃希菌属和肠杆菌属为优势属, 分别占克隆文库中总序列的46.8%和35.1%, 二者在自然界分布广泛, 大多属于人类致病菌。【结论】莫高窟第98窟壁画表面白色污染物主要为病害细菌生长所形成的菌斑群落集成。变形菌门在壁画细菌克隆文库中占绝对优势, 壁画病害微生物的出现和蔓延可能与该洞窟之前长期旅游开放存在一定关联。

关键词:敦煌壁画, 菌斑, 16S rDNA, 克隆文库, 群落组成, 结构特征, 旅游开放

Abstract:

[Objective] In this study, we aimed to observe the detailed features of microbes within white spots adhered to the surface of ancient wall paintings in Cave 98, Mogao Grottoes; meanwhile, we explore microbial community composition and structure characteristics, analyze factors that induce the explosion of microbial diseases on murals; thereafter, our results could serve with scientific support for cave conservation and tourism management. [Methods] Samples of white spots on murals were carefully collected by sterile scalpel and sealed in eppendorf tubes. A part of samples was used for observation of microbes by the scan electronic microscope (SEM); another part was used for the total genomic DNA extraction, followed steps were the bacterial 16S rDNA amplification, clone library construction, sequencing, and phylogenetic analysis, afterwards, the microbial community composition and structure characteristics were clarified. [Results] A number of objects that similar to microbes were observed in white spots of wall paintings, which shaped short rod or ovoid, with the volume ranged among 3.0 μm?5.5 μm multiply by 1.5 μm?2.5 μm. About 111 appropriate sequences were retrieved from clone library in this study. All sequences were similar to members of Enterobacteriaceae and Pseudomonadaceae that both belong to phylum of γ-proteobacteria. Results of community analysis indicated that sequences were mainly affiliated to genus Enterobacter, Escherichia, Azotobacter, Serratia, and Klebsiella; Enterobacter (46.8%) and Escherichia (35.1%) were dominant in our study, both of them are ubiquitous microbes in nature, and human pathogenic bacteria as known. [Conclusion] The white spots on murals in Cave 98 were caused by colony formation of bacterial growth. Members of proteobacteria dominated in the clone library of our study sites. As a result, we speculated that explosion and spread of microbes in Cave 98 was related to long-term tourism in this cave previously.

Key words:Dunhuang murals, Microbial colonies, 16S rDNA, Clone library, Community composition, Structure characteristics, Tourism opening