导读 | Introduction
石质文物的生物风化主要是多种微生物介导的生理生化过程共同作用的结果。当下针对石栖微生物的大量研究揭示了石质文物上的微生物群落组成与结构特征，但对于造成石质文物生物风化相关微生物组中具有活性的微生物分布特征及其代谢反应研究非常有限。本研究通过整合宏基因组和宏蛋白质组分析，确定了北石窟寺砂岩文物上微生物组中参与文物生物风化过程的关键微生物类群及其介导的生物地化过程和代谢反应特征。
The biodeterioration of stone mainly involves a combination of variety of microbially mediated physiological and biochemical activities. The previous studies on microbial community show a complex taxa composition of these stone-dwelling microbial communities but effective analysis of the distribution characteristics and viability traits of the active culprit in the microbiome responsible for the biodeterioration of stone cultural heritage is still extremely limited. This study used metagenomic and metaproteomic analysis to determine the biogeochemical processes as well as the viability traits of the active culprit for bioweathering in stone-dwelling microbiome on Beishiku Temple.
一、 石栖微生物组中的核心类群和环境敏感类群 | Stone microbiome presents of a relative stable core and a site-specific portion
北石窟寺位于中国西北地区的甘肃省庆阳市，拥有大小窟龛308个以及石雕石刻2429身。其开凿年代始于北魏年间（公元509年），止于清代。北石窟寺开凿于较为均质的山体砂岩基质上(图1)。本研究将群落组成与PATRIC（Pathosystems Resource Integration Center）生物信息资源数据库中所列生理生态类别加以整合分析，发现北石窟寺砂岩上栖生的微生物群落中既存在应对极端生存环境的核心微生物类群，也存在对特定点位上生态特征敏感的环境感受性微生物类群(图2)。
The Beishiku Temple is located at Qingyang City in Northwest China's Gansu Province. It has 308 niches and 2429 preserved stone carvings dating from the Northern Wei Dynasty (509 CE) to the Qing Dynasty . It was excavated directly into the sandstone of the mountain, and the formation of the sandstone material is relatively homogeneous (Fig. 1). To link the PATRIC (Pathosystems Resource Integration Center) categories with the taxonomic profiling, all features in the microbial composition species were grouped and assigned to obtain their common geoecological categories. Though the enrichment of selective members in microbiome takes place in response to the harsh environmental conditions on the sandstone site, there are still microbial populations sensitive to geoecological disturbances (Fig. 2).
图1 北石窟寺的整体外观及各采样区域的位置和生境特征
Fig. 1 An overall appearance and view of the Beishiku Temple with the location and environmental characteristics of each study sampling site
图2 宏基因组数据集检索到的石栖微生物群落成员的生理生态类别（PATRIC）归属及其在各采样点的丰度概况
Fig. 2 The attributed geoecological categories (PATRIC) of the stone-dwelling microbiome retrieved from the metagenome datasets and their abundance distribution among the different locations of the ancient stone monuments of Beishiku Temple
二、石质文物生物风化的关键微生物生理生化活动 | Key microbial physiological and biochemical activities in the bioweathering of stone works
宏基因组分析可全面获得微生物多样性、群落组成、生化代谢潜力特征。宏蛋白质组分析则为研究微生物群落生理代谢功能及生化过程的活力特征提供了可靠平台。本研究结果表明，宏基因组分析所获的高丰度功能类别表征微生物群落在石生环境上生存所需的必要生理能力，而宏蛋白质组分析所获的高丰度功能类别则表征群落生存面临环境压力时的生理代谢响应（图3）。
The comprehensive information on the microbial diversity, taxon composition, and biochemical potentials of the microbiome can be obtained by metagenomic analysis. Metaproteome provides vital approach for understanding microbiome functions and dynamics. Based on this study, the metagenome better reflects the basic needs of the microbiome for survival, while the metaproteome better reflects the physiological metabolic activities of the microbiome in response to environmental stresses at present (Fig. 3).
图3 石栖微生物群落的宏基因组和宏蛋白组COG功能类别注释丰度概况
Fig. 3 The ranks of Clusters of Orthologous Gene (COG) functional categories within the stone microbiome determined using multi-omics data
三、石栖微生物介导的碳固定 | Geomicrobiological carbon sequestration
本研究宏基因组数据集中，3-羟基丙酸双循环固碳途径被频繁注释，相关基因常在Chloroflexaceae科的绿色非硫细菌中发现。由于参与该固碳途径的酶均具有耐受自由氧的能力，因此北石窟寺石栖微生物群落具有好氧的生理特点。同时，宏基因组数据集中，可产生氧分子的光合固碳途径的还原性磷酸戊糖循环也被频繁注释（图4a）。除还原性乙酰-CoA固碳途径外，所有已知原核微生物和光合微生物介导的CO2固定生化途径均有被注释 (图4b)。
In our metagenome dataset, the genes involved in the 3-hydroxypropionate bicycle, which is usually found in some green non-sulfur bacteria of the family Chloroflexaceae were frequently identified. Considering that none of the enzymes involved in this cycle is inherently sensitive to oxygen, which indicates that the habitats of the indigenous microbiome should be aerobic in this study. Correspondingly, the annotated sequences affiliated with genes of the oxygen-tolerance enzymes responsible for the reductive pentose phosphate cycle in the photosynthetic carbon fixing pathway were abundant, as well (Fig. 4a). All of the known CO2 fixation pathways in prokaryotes and photosynthetic organisms were retrieved except the reductive acetyl-CoA pathway (Fig. 4b).
图4 宏基因组数据集KEGG路径重建微生物C循环功能
Fig. 4 Geomicrobiological carbon cycling functions by the KEGG pathway reconstruction of the metagenome dataset
四、石栖微生物群落介导的氮和硫循环| Nitrogen and sulfur cycling in the stone microbiome
针对本研究宏基因组数据集中检索出的微生物类属，根据微生物介导生物地化氮循环的代谢功能加以分配，找到了群落中介导生物地化氮素循环的微生物功能群，这些类群介导的代谢反应得到了宏蛋白质组分析的进一步验证（图5）。北石窟寺砂岩微生物群落具有活跃的固氮、氨氧化、硝化以及完全氨氧化代谢相互耦联过程，导致文物的生物风化。本研究发现，在北石窟寺最干燥的研究点位上（GM1），Comammox氨氧化类群表现出极高的代谢活力。
Microbial genera detected in both the stone metagenomes with an involvement in the nitrogen cycle were assigned, and a complete nitrogen cycle could be reconstructed in all sites in this study. The metabolic activity of these taxa in the microbiome was further supported by the stone metaproteome (Fig. 5). The active nitrogen fixation, ammonia oxidation, nitrification, and Comammox reaction cooperatively to initiate of biodeterioration. Furthermore, the present study indicates that the abundance of Comammox bacteria was higher in the driest site (GN1) at the expression level (proteome).
图5 利用从宏基因组和宏蛋白组数据集检索到的特定属的潜在功能，重建北石窟寺砂岩微生物组介导的N循环
Fig. 5 Reconstruction of the nitrogen cycle in the stone microbiome on Beishiku Temple using the potential capability of the specific genera retrieved from the metagenome and metaproteome datasets
在本研究检出的硫氧化（SOx）微生物类群中，Paracoccus属在宏蛋白组分析中被检出（图6），其丰度呈现出窟外环境（GD1, GM1, GU1）高于窟内环境（GN1）的特点。由此提示，北石窟寺附近石化工业产生的硫及硫氧化物的空气沉降可能为北石窟寺砂岩微生物介导的硫元素循环补充了反应底物。硫/硫氧化物的空气沉降能增强石栖微生物中SOx类群的生化代谢活力，而这一过程会加剧石质文物的酸侵蚀效应。
Among the microorganisms involved in the SOx (Sulfur-oxidizing) reaction, Paracoccus was detected in the metaproteome with a higher abundance distribution in the outdoor sites (GD1, GM1, GU1) than in the indoor sites (GN1) (Fig. 6). The results suggests that the petrochemical industry in the vicinity of Beishiku Temple may provide substrates for the sulfur cycle in the stone microbiome. The deposition of sulfur/oxidized sulfur via atmosphere way can stimulate the physiological activity of Sox, and the activity of SOx system can contribute to the acid corrosion of stone heritage.
图6 利用从宏基因组和宏蛋白组数据集检索到的特定微生物属的潜在功能重建北石窟寺砂岩微生物组介导的S循环
Fig. 6 Reconstruction of the sulfur cycle in the stone microbiome on Beishiku Temple using the potential capability of the specific genera retrieved from the metagenome and metaproteome datasets
总结 | Conclusions
本研究阐明了微生物群落应对石生环境压力时其活力响应的生理生化代谢特征，确定了完全氨氧化（Comammox）类群的代谢活力分布特点，揭示了功能微生物生理生化代谢介导的石质文物生物风化过程。与以往研究的群落组成及功能预测分析方法相比，针对微生物组中呈现活性的微生物类群及其介导的生理生化过程分析，才是研究石质文物生物风化相关功能微生物作用机制的关键。
This study clarified the physiological and biochemical metabolism of the microbial vitality response to the environmental stresses on the stone habitat. The dynamic metabolic distribution of the complete ammonia-oxidizing (Comammox) microbial taxa was then being confirmed. And revealed the processes of functional microbial mediated biochemical metabolism in the bioweathering of stone heritage. The assessment of specific metabolic reactions with the active members involved in the microbiome is a more reliable way to identify the culprits and mechanisms in bioweathering of stone monuments than the function prediction by community analysis.
扫二维码 | 查看原文
https://www.sciencedirect.com/science/article/abs/pii/S0048969723032370
本文内容来自ELSEVIER旗舰期刊Sci Total Environ第896卷发表的论文：
Wu F., Ding X., Zhang Y., Gu J-D., Liu X., Guo Q., Li J., Feng H. 2023. Metagenomic and metaproteomic insights into the microbiome and the key geobiochemical potentials on the sandstone of rock-hewn Beishiku Temple in Northwest China. Sci Total Environ, 896, 164616.
https://doi.org/10.1016/j.scitotenv.2023.164616
第一作者：武发思 研究馆员
敦煌研究院 保护研究所
敦煌研究院研究馆员，保护研究所副所长，博士。主要从事文物遗产保护生物学领域研究。现担任国际生物退化与生物降解学会（IBBS）。主持及作为骨干成员完成了包括国家自然科学基金项目、中国科学院“西部之光”人才培养引进计划项目及甘肃省科技计划项目等项目课题20余项。合作发表研究论文110余篇，参编专著3部，获授权专利10余件，获省部级科技奖励4项。
共同第一作者：丁兴华 讲师
湖南师范大学 历史文化学院
湖南师范大学历史文化学院讲师，博士。主要从事文化遗产微生物学、文化遗产保护、大型文物/大遗址的预防性保护利用等领域的研究。吴哥世界文化遗产的保护项目（JASA）参与成员，国际生物退化和生物降解协会（IBBS）会员。主持省级科研创新项目1项，参与国家863计划专项，国家自然科学基金面上项目，国家重大科学研究计划（973），Hong Kong Research Grants Council General Research Fund (GRF) Grant。发表研究论文20余篇。
共同通讯作者：顾继东 教授
广东以色列理工学院
广东以色列理工学院教授，博士生导师。获美国弗吉尼亚理工大学博士学位。曾就职于哈佛大学微生物生态实验室、香港大学生物科学学院。目前主要从事文化遗产微生物学、文化遗产保护、微生物驱动氮循环等领域研究。担任国际学术期刊International Biodeterioration & Biodegradation主编，Ecotoxicology和Journal of Polymers and the Environment 副主编。在Nature Sustainability, Trends in Ecology and Evolution，Trends in Microbiology等环境与生物技术领域高质量学术期刊发表SCI论文500余篇，入选全球前2%顶尖科学家榜单。
通讯作者：冯虎元 教授
兰州大学 生命科学学院
兰州大学生命科学学院教授，博士生导师，教育部新世纪优秀人才支持计划。主要从事菌根生物学、植物生态学及文物微生物与保护研究工作。担任甘肃省植物学会理事长。主持完成973计划子课题、国家自然科学基金项目、教育部新世纪人才计划项目、甘肃省自然科学基金等科研项目30余项。出版、编写教材及专著16部，以第一及通讯作者在New Phytologist, Global Change Biology等学术刊物上发表论文200余篇，获省部级科技奖励6项，获宝钢优秀教师奖、甘肃省“高等学校教学名师奖”。
近2年发表在Sci Total Environ上的论文：
1. Wu F., Zhang Y., Gu J-D., He D., Zhang G., Liu X., Guo Q., Cui H., Zhao J., Feng H. 2022. Community assembly, potential functions and interactions between fungi and microalgae associated with biodeterioration of sandstone at the Beishiku Temple in Northwest China. Sci Total Environ, 835, 155372.
https://doi.org/10.1016/j.scitotenv.2022.155372
2. Meng S., Qian Y., Liu X., Wang Y., Wu F., Wang W., Gu J-D. 2023. Community structures and biodeterioration processes of epilithic biofilms imply the significance of micro-environments, Sci Total Environ, 876, 162665.
https://doi.org/10.1016/j.scitotenv.2023.162665.
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