Advance Publication J. Gen. Appl. Microbiol. doi 10.2323/jgam.2017.01.007 „2017 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation Invited Review Polyphasic insights into the microbiomes of the Takamatsuzuka Tumulus and Kitora Tumulus (Received December 5, 2016; Accepted January 25, 2017; J-STAGE Advance publication date: March 24, 2017) Junta Sugiyama,1,* Tomohiko Kiyuna,2 Miyuki Nishijima,2 Kwang-Deuk An,2,# Yuka Nagatsuka,2,† Nozomi Tazato,2 Yutaka Handa,2,‡ Junko Hata-Tomita,2 Yoshinori Sato,3 Rika Kigawa,3,$ and Chie Sano3 1 TechnoSuruga Laboratory Co. Ltd., Chiba Branch Office, 3-1532-13 Hasama-cho, Funabashi, Chiba 274-0822, Japan 2 TechnoSuruga Laboratory Co. Ltd., 330 Nagasaki, Shimizu-ku, Shizuoka-city, Shizuoka 424-0065, Japan 3 Tokyo National Research Institute for Cultural Properties, 13-43 Ueno Park, Taito-ku, Tokyo 110-8713, Japan Microbial outbreaks and related biodeterioration ors. In addition, we generated microbial commu- problems have affected the 1300-year-old nity data from TT and KT samples using culture- multicolor (polychrome) mural paintings of the independent methods (molecular biological meth- special historic sites Takamatsuzuka Tumulus (TT) ods, including PCR-DGGE, clone libraries, and and Kitora Tumulus (KT). Those of TT are desig- pyrosequence analysis). These data are comprehen- nated as a national treasure. The microbiomes of sively presented, in contrast to those derived from these tumuli, both located in Asuka village, Nara, culture-dependent methods. Furthermore, the mi- Japan, are critically reviewed as the central sub- crobial communities detected using both methods ject of this report. Using culture-dependent meth- are analytically compared, and, as a result, the com- ods (conventional isolation and cultivation), we plementary roles of these methods and approaches conducted polyphasic studies of the these micro- are highlighted. In related contexts, knowledge of bial communities and identified the major micro- similar biodeterioration problems affecting other bial colonizers (Fusarium spp., Trichoderma spp., prehistoric cave paintings, mainly at Lascaux in Penicillium spp., dark Acremonium spp., novel Can- France and Altamira in Spain, are referred to and dida yeast spp., Bacillus spp., Ochrobactrum spp., commented upon. Based on substrate preferences Stenotrophomonas tumulicola, and a few (or ecological grouping) and mapping (plotting de- actinobacterial genera) and noteworthy microbial tection sites of isolates), we speculate on the possi- members (Kendrickiella phycomyces, ble origins and invasion routes whereby the major Cephalotrichum verrucisporum (∫Doratomyces microbial colonizers invaded the TT stone cham- verrucisporus), Sagenomella striatispora, ber interior. Finally, concluding remarks, lessons, Sagenomella griseoviridis, two novel and future perspectives based on our microbiologi- Cladophialophora spp., Burgoa anomala, one novel cal surveys of these ancient tumuli, and similar species Prototheca tumulicola, five novel treasures outside of Japan, are briefly presented. Gluconacetobacter spp., three novel Bordetella spp., A list of the microbial taxa that have been identi- and one novel genus and species Krasilnikoviella fied and fully or briefly described by us as known muralis) involved in the biodeterioration of mural and novel taxa for TT and KT isolates since 2008 is paintings, plaster walls, and stone chamber interi- presented in Supplementary Materials. *Corresponding author: Junta Sugiyama, TechnoSuruga Laboratory Co. Ltd., Chiba Branch Office, 3-1352-13 Hasama-cho, Funabashi, Chiba 274- 0822, Japan. Tel: +81-47-404-5738 E-mail: [email protected] #Present address: Macrogen Japan Corp., International Science Innovation Building 3F, Kyoto University, 36-1 Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan. †Present address: Mukaishima-cho, Onomichi, Hiroshima 722-0073, Japan. ‡Present address: CAF Laboratories Inc., 1257-1 Michinoue-Kannabe-cho, Fukuyama, Hiroshima 720-2104, Japan. $Present address: Kyushu National Museum, Museum Science Division, 4-7-2 Ishizaka, Dazaifu, Fukuoka 808-0118, Japan. None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work. 2 SUGIYAMA et al. Japan, 1987; Ishizaki and Kigawa, 2011), and, similarly, Key Words: bacteria; biofilm; cultural properties; for the Paleolithic caves in southern Europe, such as the culture-dependent methods; culture-independent Lascaux Cave in France, and the Altamira and other caves methods; fungi; microbial community analysis; in Spain (e.g., Coye, 2011; Saiz-Jiménez, 2014, 2015). yeasts Our collaborative research team has conducted micro- biological surveys to identify the microbiota (fungi and Abbreviations: Agency for Cultural Affairs, ACA; bacteria, in particular) involved in the biodeterioration of D1/D2 domains of nuclear large subunit ribosomal the mural paintings, plaster, stone walls and ceilings of RNA, LSU; Investigation Committee for Elucidat- both tumuli (Sugiyama et al., 2009). This work began at ing the Cause of Deterioration of the Mural Paint- the Takamatsuzuka Tumulus in 2004 at the height of the ings of the Takamatsuzuka Tumulus, serious contamination phase termed the “Explosive Ap- ICECDMPTT; = Heterotypic (taxonomic) syno- pearance of Molds in Heisei (the Second Crisis),” which nym; ∫ Homotypic (nomenclatural) synonym; was named by the Investigation Committee for Elucidat- Kitora Tumulus, KT; Takamatsuzuka Tumulus, TT ing the Cause of Deterioration of the Mural Paintings of Takamatsuzuka Tumulus (ICECDMPTT, 2010). At that time, the attention of both the scientific community (e.g., 1. Introduction ICECDMPTT, 2010) and society in general (e.g., Mohri, 2007) was focused on identifying the molds and possible Microorganisms characteristically decompose or assimi- invasion routes involved in the biodeterioration of TT late substrates, ranging from monosaccharides to the most mural paintings, such as “Asuka Beauties (Asuka Bijin)” complex polymers. Some bacteria and fungi, particularly (Fig. 1j) and “White Tiger (Byakko)”, and how to con- the basidiomycetous fungi, can decompose serve and restore them at the site. polysaccharides, cellulose, and lignin. The undesirable The original published papers and accounts based on effects of these activities, such as the destruction or de- our research are referred to, and cited, within this review. composition and spoilage of useful materials and artifacts A better understanding and elucidation of the causes and by living organisms, are categorized as biodeterioration: mechanisms involved in the biodeterioration are essential more general saprotrophic activities are categorized as for the proper conservation and restoration of mural paint- biodegradation (Deacon, 1997; Pinna and Salvadori, ings and related cultural properties. The aims of this re- 2008). Biodeterioration is simply the consequence of ex- view are to identity and describe the microorganisms in- tensive biodegradation of a material (Carlile and volved in the biodeterioration of the mural paintings and Watkinson, 1994). A variety of objects of cultural signifi- plaster walls of these national treasures. The artifacts de- cance, including mural and cave paintings, ruins, historic scribed herein were maintained in situ in a subterranean buildings, and monuments, are suitable habitats for de- environment specific to the interior of the tumulus stone structive microorganisms (e.g., Allsopp et al., 2004; chamber. This review also aims to elucidate the mecha- Caneva et al., 2008; Saiz-Jiménez, 2003, 2014, 2015; nisms involved in biodeterioration, using the insights Sterflinger, 2010). gained from data collected at the Lascaux, Altamira, and Herein, we focus mainly on the biodeterioration by mi- other caves. Covering nearly all of the data and evidence croorganisms and related consequences to the mural paint- regarding the microbiology of the Takamatsuzuka and ings, plaster, and plaster walls at two ancient Japanese Kitora Tumuli, this review is expected to provide a guide tumuli, both in Nara: the Takamatsuzuka Tumulus (TT; for future work with such artifacts so as to facilitate keep- Fig. 1a) and the Kitora Tumulus (KT; Fig. 1o). Both of ing them at the original site. these 1300-year-old tumuli contained multicolor (poly- chrome) mural paintings that were directly drawn onto thin 2. A Brief History of the Major Phases, Microbial plaster inside the respective small stone chambers. The Disturbances, and Related Biodeterioration Problems mural paintings and plaster walls of both tumuli were con- of the Paintings and Other Artifacts at Selected fined inside enclosed stone chambers under environmen- Ancient Tumuli and Caves tal conditions to be described in detail later. However, these mural paintings and plaster walls faced serious microbial The selected ancient tumuli (tombs) and caves are di- outbreaks and disturbances after respective excavations vided into four groups: TT, KT, other tumuli in Japan, and in 1972 (TT) and 2004 (KT). Eventually, the TT and its caves outside Japan. The profiles, microbial outbreaks, stone chamber were dismantled, and the 16 stone blocks, environmental conditions, and related deterioration prob- i.e., comprising eight stone (side) walls and four ceiling lems encountered at these various tumuli and caves are stones, bearing mural paintings, and four floor stones (Fig. briefly reviewed. 2), were relocated to a restoration facility in Asuka