敦煌莫高窟微生物时空分布特征及对壁画腐蚀机理研究

Mogao Caves are a site museum that combining architecture, colored sculptures, and mural paintings together, one of the world famous cultural heritage. With the changement of the natural climate and tourism ecological environment, the conservation of elegant but fragile wall paintings were under great threat. The environment in Mogao Caves may provide appropriate condition for the survive and development of microbes, which can degrade and deteriorate the pigments of wall paintings.The animal glue and vegetable gelatin used as adhesives in painting layers are both major nutrient resources for microbes. Consequently, the origin of those organisms, their invasion process, and deterioration mechanisms are major problems for microbiologists and conservators to study in Mogao Grottoes. In this research, we will target the typical caves containing wall paintings with serious microbial diseases, though the DNA extraction for bio-deteriorated wall painting samples and environmental aerial particles in typical caves of Mogao, we would construct the molecular clone library of total microbes, including bacterial and fungal groups, ascertain the community composition and structure characteristics of the total microbes, and analyze correlation of microbial structures with monitored environmental factors. After the isolating and screening of microbes in paintings, we want to discover the biological erosion progress of significant pigment (e.g., red lead and cinnabar) by microbes isolated from wall paintings, meanwhile, to clarify the biological corrosion mechanism of the wall paintings. In brief, this study would provide academic basics and technological support for the proper conservation of ancient wall paintings, and it can be a mirror for the conservation of the similar cultural relics.

敦煌莫高窟是集壁画、彩塑、建筑三位一体的遗址博物馆，著名的世界文化遗产。随着自然和旅游生态环境的变迁，精美而脆弱的壁画保存受到严重威胁。莫高窟洞窟适宜的环境条件（如温湿度）和制作壁画时使用的有机粘合剂均为病害微生物的生长和繁殖提供了有利条件。有害微生物的来源、入侵途径、腐蚀机理研究则是莫高窟壁画保护面临的主要问题之一。本研究拟选择莫高窟典型洞窟，通过提取壁画表面和大气中微生物总DNA，构建分子克隆文库，确定腐蚀壁画微生物群落组成，探究其与环境因子间相互关系；通过分离、筛选腐蚀壁画微生物菌株，采用模拟实验，揭示壁画特征颜料（如铅丹、朱砂）受功能型微生物腐蚀的生物学过程，阐明微生物腐蚀壁画的内在机理，为敦煌壁画有效保护提供理论依据和技术支撑，为同类文物保护提供借鉴。

微生物腐蚀退化是威胁古代壁画长久保存的关键因素之一。本项目研究了敦煌莫高窟壁画微生物与空气微生物的群落组成和结构特征，解析了环境温湿度、降雨量、游客数量等因子对文物赋存环境微生物的影响，揭示了优势菌株及其代谢产物对壁画特征颜料的腐蚀机理。结果表明，莫高窟第53、54、98窟壁画微生物病害比例分别为26.35%、4.87%和9.44%。克隆文库和454高通量测序分析显示，第53窟壁画菌斑中草酸杆菌属（21.4%)、假洛卡氏菌属（13.1%）和乳酸球菌属（10.9%）为优势属；而乳酸球菌属（62.1%）和假单胞菌属（10.1%）在无菌斑壁画中占优势。血球丛赤壳属（36.9%）和枝孢属（13.4%）是菌斑优势真菌属，而久浩酵母（16.5%）、血球丛赤壳属（12.8%）和耐碱酵母属（12.1%）在无菌斑壁画中占优势。第54窟菌斑壁画中以变形菌门、厚壁菌门、放线菌门为主，无菌斑壁画中厚壁菌门、变形菌门和拟杆菌门占优势，多数真菌在属水平上分类地位未确定。第98窟壁画白色污染物生长集聚形成菌斑，与人类活动密切相关的埃希菌属（46.8%）和肠杆菌属（35.1%）为优势属。第251窟壁画棕黑色斑点为昆虫活动及排泄物集聚形成。对比壁画表面、地仗层和降尘样品中菌群特征发现，壁画土著菌和外源性空气颗粒物沉降是其病害菌的重要来源。环境空气颗粒物中微生物多样性、主成分和聚类分析表明，窟内外PM2.5间真菌群落组成相似，与PM10-2.5差异较大，而细菌群落为窟内与窟外PM10-2.5中相似。窟内微生物浓度、空气颗粒物浓度、温度、RH和CO2浓度变化受游客参观的影响较大，洞窟微环境在参观结束5-6h后才基本恢复到初始状态。第53、54窟内RH在监测期均低于75%，可抑制病害菌活动，但第98窟窟顶最大RH达80.39%，主要受到窟外阴雨天气影响，高湿度易诱发壁画菌害和盐害。GC-MS检测到壁画中角鲨烯和脂肪酸等微生物代谢产物。模拟实验显示，颜料抗性菌株黑曲霉、产黄青霉等通过代谢产生草酸、醋酸等有机酸与铅丹反应，形成草酸铅(PbC2O4)、醋酸铅((CH3COO)2Pb)等有机盐。少动鞘氨醇单胞菌对铅丹具有吸附或转化作用，并可能通过念珠藻黄素等代谢产物的沉积，形成棕褐色菌体污染壁画。本研究阐明了基于微生物产酸和色素沉积的壁画腐蚀机理，项目成果为莫高窟及同类文化遗产的有效保护与合理开放提供了科学依据。