四川锦屏隧道细菌群落特征及其组装过程

BACTERIAL COMMUNITY AND THEIR ASSEMBLY PROCESS IN JINPING TUNNEL,SICHUAN PROVINCE

深地生物圈微生物群落组成及其潜在功能研究对于探索生命生存极限,安全开发利用地下空间具有重大意义。锦屏隧道位于四川省凉山彝族自治州,上覆地层为三叠纪的大理岩和灰岩,其最大埋深可达2525 m,该隧道的开挖为研究陆地深地生物圈微生物群落提供了宝贵的机会。为了探究锦屏隧道细菌群落组成、潜在功能及群落组装机制,分别采集锦屏隧道内松散的表层岩壁和沉积物样本进行16S rRNA扩增子的高通量测序分析。结果表明锦屏隧道细菌群落具有较高的多样性,香农威纳指数变化范围为5.19~8.71。群落组成上变形菌门(Proteobacteria)在所有样本中占主导地位,其次为放线菌门(Actinobacteria)和浮霉菌门(Planctomycetes)。纲水平上,γ-变形菌纲(Gammaproteobacteria)占绝对主导,在大部分样本中的相对丰度超过61.4%,α-变形菌纲(Alphaproteobacteria)和β-变形菌纲(Betaproteobacteria)也有较高的相对丰度。代谢功能上,锦屏隧道中化能异养细菌类群在锦屏隧道中占绝对主导,而以H2和CH_(4)为能源进行代谢的细菌类群相对丰度极低,经岩溶裂隙进入隧道内的地表有机质是这些异养细菌类群的潜在物质和能量来源。此外,与深地基岩和地表土壤环境研究结果对比发现,锦屏隧道内动物寄生及人类潜在病原体细菌类群的相对丰度较高(分别达到6.4%和6.3%),这表明深地生物圈潜在的生物安全隐患及人类活动对深地生物圈的扰动不可忽视。锦屏隧道细菌群落的共发生网络具有良好的模块性,Ralstonia、Planctomyces和Woodsholea是维持网络结构稳定的关键类群。群落构建分析表明锦屏隧道细菌群落构建以确定性过程为主导,其中异质性选择贡献最大,在岩壁微生物群落构建中占52.8%,对沉积物微生物群落构建以及对所有样本微生物群落构建的贡献分别为61.1%和61.4%,这可能是由锦屏隧道黑暗无光且寡营养的极端环境特点所决定。该研究结果揭示了四川锦屏隧道深地生物圈细菌的群落组成以及化能异养为主的生态功能特征,明确了环境条件对细菌群落构建的决定性作用,也为更好地开发利用锦屏地下空间提供了重要的生物安全参考。

Understanding of the composition and potential function of microbial communities in deep biosphere is of great significance,which benefits for the understanding of life limit and offers valuable information on bio-safety in utilization of underground space.Jinping Tunnel(28°20'50″N,101°56'0″E)is located in Liangshan Yi Autonomous Prefecture,Sichuan Province.The overlying rock is Triassic marble and limestone,and the maximum buried depth can reach 2525 m.Bacterial community plays an important role in the ecological function of deep biosphere,the excavation of Jinping tunnel provides a valuable opportunity for the study of microbial community in the terrestrial deep biosphere.To explore the bacterial composition,potential functions and assembly mechanism in Jinping tunnel,loose surface rock wall and sediment samples were collected and subjected to 16S rRNA high-throughput sequencing.The Shannon Wiener index of bacterial communities in Jinping tunnel ranged from 5.19 to 8.71,which were higher than those previously reported from other deep biosphere study.Proteobacteria dominated bacterial communities in all samples,followed by Actinobacteria and Planctomycetes.At the class level,Gammaproteobacteria was the dominant group,with relative abundance over 61.4%in most samples.Alphaproteobacteria and Betaproteobacteria also showed high relative abundance.In terms of metabolic functions,heterotrophic bacterial groups were dominant,whereas bacterial groups using H2 and CH_(4)were in minor portions.The input of surface organic matter with water through karst fissures might provide substrate for these heterotrophs.Compared with microbial functions in deep bedrocks and surface soils,relatively high abundances of potential animal parasitism(6.4%)and human potential pathogens(6.3%)were surprisingly found in the functional annotations in Jinping tunnel,which may suggest the potential biosafety risks and human disturbance to the deep biosphere during the construction of the tunnels.The occurrence network analysis indicated a tightly connected modularity structure with more positive links among different modules and with Ralstonia、Planctomyces and Woodsholea as the keystone species.Community assembly analysis showed that the bacterial community assembly was dominated by deterministic processes,among which variable selection contributed the most,accounting for 52.8%,61.1%and 61.4%in the tunnel wall,sediments and all samples,respectively,in Jinping tunnel.This may result from the extreme environmental conditions,which was characterized by darkness and oligotrophic conditions.Our results were the first report about the bacterial biota in Jinping tunnel,which provided the valuable information about bacterial community composition,potential functions and confirmed the role of environmental selection on bacterial community assembly.It not only enhanced our understanding about the terrestrial deep biosphere,but also provided important biosafety references for better development and utilization of Jinping underground space.