Isolation and diversity of culturable anaerobes from the ivory burial environment of Sanxingdui

Abstract:

[Background] The deterioration of cultural relics in burial environments is closely associated with microorganisms that are recognized as one of the major contributors to the degradation and disappearance of cultural relics. The knowledge is limited regarding anaerobes in the anoxic conditions deep at the Sanxingdui site. [Objective] To investigate the culturable anaerobe diversity in the ivory burial layer of Sanxingdui, explore the factors influencing the distribution of culturable anaerobes, and provide strain resources and theoretical reference for further research on the organic acid and protease production of the anaerobes and the mechanism of ivory degradation. [Methods] Anaerobes were isolated from the ivory burial layer of Sanxingdui using an anaerobic workstation and three isolation media, and the effectiveness of different media for isolating anaerobes was analyzed. The phylogenetic analysis based on 16S rRNA gene sequences was performed to reveal the diversity of culturable anaerobes. Furthermore, the Mantel test was carried out to study the environmental factors influencing the distribution of culturable anaerobes. [Results] A total of 61 strains of anaerobes were isolated from the ivory burial layer of Sanxingdui. Based on the 16S rRNA gene sequences, the strains were assigned into Clostridium, Terrisporobacter, Hathewaya, and Haloimpatiens belonging to Clostridiaceae and Peptostreptococcaceae. Eleven species were identified: Clostridium cibarium, C. beijerinckii, C. magnum, C. nitrophenolicum, C. kogasense, C. intestinale, C. sporogenes, Clostridium sp. (A4d), Terrisporobacter mayombei, Hathewaya massiliensis, and Haloimpatiens massiliensis. Among the three isolation media used, the reinforced Clostridium medium yielded the highest number and the most species of fermentative anaerobes, and the other two media also showed unique isolation effects. Mantel test results indicated significant positive correlations between the distribution characteristics of culturable fermentative anaerobes and the content of Cu and Pb. [Conclusion] The culturable fermentative anaerobes present varied diversity in the ivory burial environment of Sanxingdui, with the highest richness and diversity in pit K8. Multiple isolation media facilitated the isolation of diverse microbial groups. Cu, Pb, and organic matter are the key factors influencing the distribution of culturable fermentative anaerobes in the ivory burial layer of Sanxingdui.