Cryo-micro-Raman spectroscopy and next generation sequencing for investigation of biomarkers in water ice inclusions of the subglacial antarctic lake

该项目的目的是发现沃斯托克湖上栖息的微生物，无论是现存的还是灭绝的。将评估低温拉曼显微镜和下一代测序(NGS)在准确检测和识别特定生物来源标记方面的科学潜力，并将其应用于鉴定从冰下南极沃斯托克湖获得的水冰芯中的有机物相关物质。随后的两种分析技术将应用于冰冻样品，首先是不熔化的样品，然后是熔化的产品。首先，微拉曼光谱将能够实现与有机物相关的非接触式定位和识别，防止被困在冰基质中的固体和气体中的物质受到污染和蒸发。拉曼光谱将应用于天然和人造冰样，以及稍后从沃斯托克湖冰中提取的固体包裹体。有机碳、脂类和蛋白质作为DNA的替代物，被选为拉曼光谱的可能的特异性生物标志物，用于这些原始样品的拉曼光谱，这些样品中含有非常罕见的固体和一些挥发性气体包裹体。人工生物标志物-冰样的检测下限将在项目框架内确定。在给出正反馈的情况下，冰可以在实验室的洁净室条件下融化，固体包裹体(以及局部融化)可以通过拉曼光谱和互补的生物地球化学方法进行重新研究。提取的微生物基因组DNA的下一代测序以及补充分析(微粒计数、扫描电子显微镜和原子力显微镜、有机碳含量、分析水化学、细胞浓度计数)将用于后处理样本。PCR和NGS测序将允许证明拉曼结果，并发现沃斯托克湖栖息着微生物，无论是现存的还是灭绝的。这项研究将对太空研究和天体生物学产生影响。具有用于原地行星探索的有效载荷的空间飞行任务(例如拉曼光谱仪)将搜索太阳系天体上的生命，其中海洋被冰壳覆盖，这在某种程度上可以与沃斯托克湖相媲美。作为天体生物学的主要目标，寻找现存或灭绝的地外生命是基于生物标志物的揭示和表征。寻找这种生命特征的主要对象是冰冻的卫星和以极地冰盖为特征的行星，例如火星上富含冰水的地下冻土层，以及木星的欧罗巴和土星的土卫二上被冰覆盖的海洋。预计要研究的样品是包含不同包裹体的水冰，这些包裹体嵌入到只对轻气体(氦、氢)可渗透的坚韧晶格中。冰下的南极沃斯托克湖是一个独特的陆地类比。该项目旨在加强积极参与极地区域研究和空间研究/天体生物学的研究小组之间的欧洲国际合作。

The aim of the project is to discover Lake Vostok inhabiting microorganisms, extant or extinct. The scientific potential of cryo Raman microscopy and Next Generation Sequencing (NGS) for accurate detection and identification of specific biogenic markers will be evaluated and applied for identification of organic-matter related substances in the water ice cores obtained from the subglacial Antarctic Lake Vostok. Two consequent analytical techniques will be applied to the icy samples, first without their melting and after that on the melted products. First, micro-Raman spectroscopy will enable contactless localization and identification of organic matter related substances, preventing contamination and evaporation of material entrapped in an ice matrix, solid and gaseous. Raman spectroscopy will be applied on both natural as well as artificial ice samples, and later on solid inclusions recovered from the Lake Vostok ice. The organic carbon, lipids and proteins as DNA proxy have been selected as probable specific biomarkers for Raman spectroscopy for these pristine samples with very rare solid and some volatile gas inclusions. The limits of detection for the artificial biomarker-ice samples will be determined in the frame of the project. Given a positive feedback the ice can be melted in the laboratory under clean room conditions and a solid inclusion (along with the local melt) can be reinvestigated by Raman spectroscopy along with complementary biogeochemical methods. The PCR followed by Next Generation Sequencing of extracted microbial genomic DNA along with complementary analyses (microparticle counting, SEM and AFM, organic carbon content, analytical water chemistry, cell concentration counting) will be used for the post-treated samples. The PCR and NGS sequencing will allow proving the Raman results and discover Lake Vostok inhabiting microorganisms, extant or extinct. The research will have impact on space research and astrobiology. Space missions with payloads for in-situ planetary exploration (e.g. Raman spectrometers) will search for life on Solar system bodies with oceans which are covered by ice crusts are somewhat comparable to the Lake Vostok. The search for extant or extinct extraterrestrial life as a main goal of astrobiology is based on revealing and characterization of biomarkers. The main objects to search for such life signatures are icy moons and planets featured by polar ice caps, e.g. frozen-water-rich subsurface permafrost on Mars and ice-covered oceans on Jupiter’s Europa and Saturn’s Enceladus. The samples to study are expected to be water ice containing different inclusions embedded into a tough crystalline lattice permeable for light gases (helium, hydrogen) only. The subglacial Antarctic Lake Vostok serves as a unique terrestrial analogue. The project aims to strengthen the European international cooperation between research groups actively participating in polar region research and in space research/astrobiology.