Characterization of black patina from the Tiber River embankments using Next-Generation Sequencing

Black patinas are very common biological deterioration phenomena on lapideous artworks in outdoor environments. These substrates, exposed to sunlight, and atmospheric and environmental agents (i.e. wind and temperature changes), represent extreme environments that can only be colonized by highly versatile and adaptable microorganisms. Black patinas comprise a wide variety of microorganisms, but the morphological plasticity of most of these microorganisms hinders their identification by optical microscopy. This study used Next-Generation Sequencing (NGS) (including shotgun and amplicon sequencing) to characterize the black patina of the travertine embankments (muraglioni) of the Tiber River in Rome (Italy). Overall, the sequencing highlighted the rich diversity of bacterial and fungal communities and allowed the identification of more than one hundred taxa. NGS confirmed the relevance of coccoid and filamentous cyanobacteria observed by optical microscopy and revealed an informative landscape of the fungal community underlining the presence of microcolonial fungi and phylloplane yeasts. For the first time high-throughput sequencing allowed the exploration of the expansive diversity of bacteria in black patina, which has so far been overlooked in routine analyses. Furthermore, the identification of euendolithic microorganisms and weathering agents underlines the biodegradative role of black patina, which has often been underestimated. Therefore, the use of NGS to characterize black patinas could be useful in choosing appropriate conservation treatments and in the monitoring of stone colonization after the restoration interventions.黑色生物结皮（Black patinas）是户外石质文物上极为常见的生物劣化现象。这类暴露于日光、大气及环境因子（如风力与温度波动）的石质基底，属于极端生境，仅能由适应性强、功能多样的微生物定植。黑色生物结皮包含丰富的微生物类群，但多数微生物的形态可塑性极强，导致光学显微镜难以实现精准鉴定。本研究采用下一代测序技术（Next-Generation Sequencing，NGS，涵盖鸟枪法测序与扩增子测序），对意大利罗马台伯河沿岸石灰华堤岸（muraglioni）上的黑色生物结皮开展群落解析。整体测序结果揭示了细菌与真菌群落的高度多样性，共鉴定出百余个分类单元。NGS验证了光学显微镜下观测到的球状与丝状蓝细菌的生态重要性，并揭示了真菌群落的完整分布特征，明确了微菌落真菌与叶际酵母菌的存在。本研究首次借助高通量测序技术，探明了黑色生物结皮中细菌的广泛多样性——这一多样性在以往常规分析中始终被忽视。此外，研究鉴定出的石内微生物与风化因子，进一步证实了黑色生物结皮的生物降解作用，而该作用此前常被低估。因此，利用NGS技术解析黑色生物结皮，可为筛选适宜的文物保护处理方案，以及修复干预后石质基底的微生物定植监测提供重要参考。