Polyphasic insights into the microbiomes of the Takamatsuzuka Tumulus and Kitora Tumulus

Abstract

Microbial outbreaks and related biodeterioration problems have affected the 1300-year-old multicolor (polychrome) mural paintings of the special historic sites Takamatsuzuka Tumulus (TT) and Kitora Tumulus (KT). Those of TT are designated as a national treasure. The microbiomes of these tumuli, both located in Asuka village, Nara, Japan, are critically reviewed as the central subject of this report. Using culture-dependent methods (conventional isolation and cultivation), we conducted polyphasic studies of the these microbial communities and identified the major microbial colonizers (Fusarium spp., Trichoderma spp., Penicillium spp., dark Acremonium spp., novel Candida yeast spp., Bacillus spp., Ochrobactrum spp., Stenotrophomonas tumulicola, and a few actinobacterial genera) and noteworthy microbial members (Kendrickiella phycomyces, Cephalotrichum verrucisporum (∫ ∫ ∫ ∫ ∫Doratomyces verrucisporus), Sagenomella striatispora, Sagenomella griseoviridis, two novel Cladophialophora spp., Burgoa anomala, one novel species Prototheca tumulicola, five novel Gluconacetobacter spp., three novel Bordetella spp., and one novel genus and species Krasilnikoviella muralis) involved in the biodeterioration of mural paintings, plaster walls, and stone chamber interiors. In addition, we generated microbial community data from TT and KT samples using cultureindependent methods (molecular biological methods, including PCR-DGGE, clone libraries, and pyrosequence analysis). These data are comprehensively presented, in contrast to those derived from culture-dependent methods. Furthermore, the microbial communities detected using both methods are analytically compared, and, as a result, the complementary roles of these methods and approaches are highlighted. In related contexts, knowledge of similar biodeterioration problems affecting other prehistoric cave paintings, mainly at Lascaux in France and Altamira in Spain, are referred to and commented upon. Based on substrate preferences (or ecological grouping) and mapping (plotting detection sites of isolates), we speculate on the possible origins and invasion routes whereby the major microbial colonizers invaded the TT stone chamber interior. Finally, concluding remarks, lessons, and future perspectives based on our microbiological surveys of these ancient tumuli, and similar treasures outside of Japan, are briefly presented. A list of the microbial taxa that have been identified and fully or briefly described by us as known and novel taxa for TT and KT isolates since 2008 is presented in Supplementary Materials. 64SUGIYAMA et al.

Stenotrophomonas tumulicola sp. nov., a major contaminant of the stone chamber interior in the Takamatsuzuka Tumulus

“…TT andKT were excavated in 1972 and2004, respectively, and Since the respective excavations, both stone chamber interiors have been exposed to microbial disturbances (e.g. moulds and bacteria) that eventually produced biofilms (or viscous gels) on the wall plaster of the chamber interiors (Kigawa et al, , 2015Sugiyama et al, 2009;Ishizaki & Kigawa, 2011). The stone walls of TT and KT have been relocated and the mural paintings are being repaired.…”

“…Sugiyama et al, 2009;Tazato et al, 2012;Nishijima et al, 2013). We obtained 365 bacterial isolates from 64 samples of TT and 42 samples of KT collected between September 2005 and April 2007 ) and September 2005 and June 2006, respectively.…”

Yamadazyma kitorensis f.a., sp. nov. and Zygoascus biomembranicola f.a., sp. nov., novel yeasts from the stone chamber interior of the Kitora tumulus, and five novel combinations in Yamadazyma and Zygoascus for species of Candida

“…The sampling of materials and isolation of microorganisms were performed as described in our previous studies (Kiyuna et al, 2008;Nagatsuka et al, 2009;Sugiyama et al, 2009 Y. Nagatsuka and others region including the 5.8S rRNA gene were reported previously (Yamada et al, 1999;Nagatsuka et al, 2002Nagatsuka et al, , 2005. Amplification and sequencing of MtSm and the COX2 gene were carried out as reported by Kurtzman & Robnett (2003), using primers MS-1 and MS-2 for MtSm and primers COII-5 and COII-3 for COX2.…”

“…Subsequently, until the end of 2013, the stone chamber was backfilled. Overviews of the serious microbial outbreaks that occurred inside the KT stone chamber over the 10 years from 2004 to 2013, along with the protective measures taken in response, have been provided by Kigawa et al (2009bKigawa et al ( , 2013Kigawa et al ( , 2015, Sugiyama et al (2009) and Ishizaki & Kigawa (2011).…”

Art‐omics: multi‐omics meet archaeology and art conservation

“…In Nara, Japan, Sugiyama et al . () used a combination of culture‐dependent and culture‐independent techniques to study some of the 1300‐year‐old polychrome mural paintings of the historical sites of Takamatsuzuka and Kitora Tumulus, which resulted in the identification of a new bacterial species of the Stenotrophomonas genus (Handa et al ., ). Interestingly, severe outbreaks of microbial contaminants were reported, involving genera such as Doratomyces sp.…”