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栖息的微生物，现存或灭绝。将评估低温拉曼显微镜和下一代测序（NGS）用于准确检测和识别特定生物标记的科学潜力，并将其应用于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的识别。两种随后的分析技术将应用于冰样品，首先是不融化，然后是融化的产品。首先，显微拉曼光谱将能够无接触地定位和识别有机物质相关物质，防止固体和气体冰基质中捕获的材料的污染和蒸发。拉曼光谱将应用于天然和人工冰样品，后来从东方湖冰回收的固体夹杂物。有机碳、脂类和蛋白质作为DNA代用品，被选为这些具有非常罕见的固体和一些挥发性气体包裹体的原始样品的拉曼光谱的可能的特异性生物标志物。人造人造冰柱-冰样品的检测极限将在项目框架内确定。给出正反馈，可以在实验室中在洁净室条件下融化冰，并且可以通过拉曼光谱沿着补充的地球化学方法重新研究固体包裹体（沿着局部融化）。对提取的微生物基因组DNA进行PCR，然后进行沿着下一代测序，并进行补充分析（微粒计数、SEM和AFM、有机碳含量、分析水化学、细胞浓度计数），用于后处理样品。PCR和NGS测序将允许证明拉曼结果，并发现沃斯托克湖栖息的微生物，现存或灭绝。这项研究将对太空研究和天体生物学产生影响。携带有效载荷进行实地行星探索的空间飞行任务（例如拉曼光谱仪）将在太阳系天体上寻找生命，其海洋被冰壳覆盖，有点类似于东方湖。寻找现存或灭绝的外星生命是天体生物学的主要目标，其基础是揭示和表征生物标志物。寻找这种生命特征的主要对象是冰冷的卫星和极地冰盖的行星，例如火星上富含冷冻水的地下永久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。研究的样品预计是水冰，其中包含嵌入到坚韧晶格中的不同内含物，仅可渗透轻气体（氦，氢）。南极洲冰下湖沃斯托克是一个独特的陆地模拟。该项目旨在加强积极参与极地研究和空间研究/天体生物学的研究团体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.