The community distribution of bacteria and fungi on ancient wall paintings of the Mogao Grottoes

OPEN SUBJECT AREAS: APPLIED MICROBIOLOGY BIODIVERSITY Received 23 May 2014 Accepted 21 November 2014 Published 13 January 2015 Correspondence and requests for materials should be addressed to H.F. (. cn) or W.W. (wwanfu@hotmail. com) The community distribution of bacteria and fungi on ancient wall paintings of the Mogao Grottoes Yantian Ma1, He Zhang1, Ye Du1, Tian Tian1, Ting Xiang1, Xiande Liu1, Fasi Wu2, Lizhe An1, Wanfu Wang2, Ji-Dong Gu3 & Huyuan Feng1 1 School of Life Sciences, MOE Key Laboratory of Cell Activities and Stress Adaptations, Lanzhou University, Lanzhou, Gansu 730000, People’s Republic of China, 2The Conservation Research Institute of Dunhuang Academy, Dunhuang, Gansu 736200, People’s Republic of China, 3Laboratory of Environmental Microbiology and Toxicology, School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, People’s Republic of China. In this study, we compared the microbial communities colonising ancient cave wall paintings of the Mogao Grottoes exhibiting signs of biodeterioration. Ten samples were collected from five different caves built during different time periods and analysed using culture-independent and culture-dependent methods. The clone library results revealed high microbial diversity, including the bacterial groups Firmicutes, Proteobacteria, Actinobacteria, Acidobacteria, Cyanobacteria, Bacteroidetes, Gemmatimonadetes, Planctomycetes, and Chloroflexi and the fungal groups Euascomycetes, Dothideomycetes, Eurotiomycetes, Sordariomycetes, Saccharomycetes, Plectomycetes, Pezizomycetes, Zygomycota, and Basidiomycota. The bacterial community structures differed among the samples, with no consistent temporal or spatial trends. However, the fungal community diversity index correlated with the building time of the caves independent of environmental factors (e.g., temperature or relative humidity). The enrichment cultures revealed that many culturable strains were highly resistant to various stresses and thus may be responsible for the damage to cave paintings in the Mogao Grottoes. B iodeterioration is often observed on items of cultural heritage, historic artefacts and monuments, and even for modern materials, buildings, museums and private collections1–5. Microorganisms can colonise the surfaces of a wide range of materials and niche types in ecosystems; thus, biodeterioration is observed ubiquitously. The biodeterioration of precious and culturally significant items is undesirable and a public concern. Many studies have demonstrated extensive growth of various microorganisms and resultant stains and spots on painting surfaces6,7. Some novel microbial species have been associated with the deterioration of rock paintings using culture-dependent methods, and high microbial diversity has been observed in different environments based on rRNA gene-PCR amplification and sequencing methods8,9. Although distinguishing the functional groups of microorganisms within the microbial communities of different environments is difficult, a common, core microbial group has been associated with biodeterioration in caves with similar climate conditions10. The Mogao Grottoes are located 25 km southeast of Dunhuang City in Gansu Province, China. Dunhuang City is a northwest oasis city located in the western Hexi Corridor and was an important strategic location on the ancient Silk Road connecting Europe and Asia. The caves of the Mogao Grottoes were built from the Northern Wei (386-534 AD) to the Mongolian-ruled Yuan Dynasty (1276-1368 AD) over a period of approximately 1,000 years, with more than 700 caves built and nearly 45,000 square meters of mural paintings completed. A total of 452 caves have survived the damage inflicted by nature and humans. The Mogao Grottoes are a famous world cultural heritage site known for its numerous caves, mural areas, and documents and its long time span. The Mogao Grottoes were added to the World Heritage List in 1987 as the only site that satisfied all six criteria for inclusion; a site must meet at least one requirement for inclusion on the World Heritage List11. The Mogao Grottoes are inland and are surrounded by desert, including the Gobi desert of Mongolia to the northeast and the Taklimakan desert to the northwest. It has an arid continental climate with an average annual temperature of 10.9uC, average annual relative humidity of 28.5%, yearly rainfall of 39.9 mm, and mean evaporation of 2,490 mm. These dry climate conditions limit the proliferation of most microorganisms and, consequently, many of the organic materials used in the mural paintings have resisted significant damage or deterioration. Although the remote location and arid climate ensured that the Mogao Grottoes remained, visible SCIENTIFIC REPORTS | 5 : 7752 | DOI: 10.1038/srep07752 1 www.nature.com/scientificreports discoloration and damage have accumulated over a long period of time. Large areas of the mural paintings exhibit visible signs of decay due to salt precipitation and subsequent flaking, powdering or delamination. Damage due to pigmentation and discoloration induced or caused by microbial contamination, colonisation and growth is also prevalent (Figure S1). Several methods of desalination to control salt damage of mural paintings have been evaluated with promising results and are now widely used. Although the microbial biodeterioration of Dunhuang mural paintings has been studied, efficient, effective, safe, reliable measures have not been developed12. Biocides were once frequently used to combat biodeterioration but have since been shown to be effective for only a short period of time5,13. Research is now focused on analysing the characteristics of microbial community composition and structure related to biodeterioration. The microbial population on Dunhuang mural paintings was first characterised in the 1990s, providing primary insights into the microbes associated with mural decay. The culturable microorganisms from 51 discoloured samples from 6 ancient caves were identified, and several genera including Aspergillus, Cladosporium and Flavobacterium, were associated with accelerated ageing of cementing materials and the discoloration of mural paintings14. Subsequent experiments in which simulated mural paintings were inoculated with the isolated microbial strains revealed that microbial metabolites (e.g., pigments and oxalate) altered the crystal shape of the paints and the chemical valence state of the metallic elements in the paints. These processes played an important role in the discoloration of the mural paintings, particularly for red pigment containing red lead (lead tetroxide)15. Aerial microorganisms and their seasonal dynamics were recently investigated both inside and outside of the caves at the Mogao Grottoes. The influence of tourists and environmental factors were identified as the main factors contributing to biodeterioration16,17. In addition, microbial species with the potenti (...truncated)