三星堆象牙埋藏环境可培养厌氧微生物分离及多样性分析

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

摘要:

【背景】埋藏环境与文物劣化有着密切的关系，微生物被认为是导致文物劣化和消失的重要原因之一。埋藏环境下的三星堆象牙处于深埋、缺氧的条件，但目前对该环境下的厌氧菌缺乏必要的了解。【目的】以三星堆象牙埋藏层土壤为研究对象，探究埋藏环境可培养厌氧微生物多样性和影响微生物分布的因素，为深入研究厌氧微生物产酸、产蛋白酶等功能及象牙劣化机理提供菌株资源和理论参考。【方法】利用厌氧工作站，采用3种厌氧菌分离培养基分离三星堆象牙埋藏层土壤中的厌氧微生物，分析不同培养基对厌氧菌的分离效果，对分离菌株进行16S rRNA基因系统发育分析，揭示三星堆不同象牙埋藏环境中可培养厌氧微生物的多样性，并采用Mantel检验研究影响三星堆象牙埋藏环境可培养厌氧微生物分布的环境因子。【结果】从三星堆象牙埋藏层土壤中共分离获得61株厌氧菌，16S rRNA基因测序分析结果显示分离菌株均属于发酵厌氧微生物，分属于梭菌科(Clostridiaceae)与消化链球菌科(Peptostreptococcaceae)共2个科，梭菌属(Clostridium)、土生孢杆菌属(Terrisporobacter)、哈撒韦氏菌属(Hathewaya)、Haloimpatien共4个属，Clostridium cibarium、拜氏梭菌(Clostridium beijerinckii)、大梭菌(Clostridium magnum)、硝醛酚梭菌(Clostridium nitrophenolicum)、Clostridium kogasense、肠梭菌(Clostridium intestinale)、生孢梭菌(Clostridium sporogenes)、Clostridium sp. (A4d)、马永贝土生孢杆菌(Terrisporobacter mayombei)、Hathewaya massiliensis、Haloimpatiens massiliensis共11个种；所采用的3种分离培养基中，强化梭菌培养基分离获得的发酵厌氧菌数量和种类最多，但其他2种培养基也表现出独特的分离效果；Mantel检验结果表明，可培养发酵厌氧菌种群分布特征与Cu、Pb的含量呈显著正相关。【结论】三星堆象牙埋藏环境可培养发酵厌氧微生物的分布存在一定差异，K8象牙埋藏层土壤中可培养发酵厌氧菌丰富度与多样性均最高，多种培养基综合利用有利于分离出更为丰富多样的微生物类群，Cu、Pb与有机质含量是影响三星堆象牙埋藏层土壤可培养发酵厌氧菌种群分布的关键因素。

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.