ERA-netLAC Cave ice microbiom: metabolic diversity and activity in response to climate dynamics and anthropogenic pollution CAVICE (DCC-0178

Ice caves represent highly preserved and relatively unexposed ice environments constituting a unique archive for studying the impact of climate change and anthropogenic pollution on the microorganisms living there. Still there is only scarce information about the indigenous ice-microbiota, and their vulnerability to climate change. It is only for the last two decades that it was confirmed that such glacial systems could be livable habitats for microorganisms. Ice covers about 10% of the Earth's surface. Most of the glaciers are under great pressure from the antropogenic caused climate changes, and are about to melt away, before we get knowledge about which organisms exist inside the ice, how they are able to live there and which ecosystem processes they contribute to. We currently know very little about the ice-dwelling microbes and how they will react to the globally increasing temperature. This is an ERANet-Lac project that cooperates with Romania, Chile and Argentina, and at the end of the project we will do comparative analyses on the data obtained from the different biogeographical sites. In this project, we will study the total and active microbial communities from ice caves of different climate and pollution exposures in order to understand the microbial diversity and metabolisms along a Chrono sequence model reflecting the impact of both climate and environmental pollution on cave ice microbioms. In our part of this project we will focus on the Svarthamar ice cave in Norway, which is a perennial ice block harbored within a karst cave. The Svarthamar ice cave has four entrances, two large and two small. It represents a dynamic ice cave, possessing the largest cave room in Norway and one of the lowest altitude ice caves in Europe. Radiocarbon dating of plant debris at the base of the ice indicates that ice accumulation commenced after AD 1200 (Lauritzen et al. 2005). In October 2016, a research team of nine scientists joined the field campaign in Svarthamar ice cave close to Bodø in Northern Norway. Six of the participants were from the Norwegian partner at the University of Bergen and three from the Romanian partner. The work inside the cave was carried out for 4 days. As the entire Chrono sequence is exposed inside the cave, ice cores were drilled and samples covering the entire time span. More than 500 samples were collected along the timespan for isotope analyses and reconstruction of climate changes, and seven sites were selected along the chronosequence for an in depth study with 14C dating and metagenomics analyses. The ice cores were transported frozen back to University of Bergen and processed further in the laboratory. Pollen analyses were performed and we found the samples to have input from trees and shrubs, herbs and ferns. The microbial biomass was collected and DNA and RNA were extracted. The functional variability of total and active-embedded microbiome have been targeted by direct sequencing using new sequencing technology (Illumina) of metagenomes and metatranscriptome from the ice. We could see different microbial community compositions in the various samples and there were also some variation between the DNA and RNA indicating that there indeed is a functional active part of the community living and thriving in these old, cold and dark ice systems. The data that we have obtained will be compared with other ice and glacier caves processed within the project. Further analyses in order to gain insight into the microbial community structure and metabolism will be achieved through the reconstruction of the microbial transcriptome and genome resources of the entire microbial community. In the project, we will measure the effect of past and present climate variations on the functional diversity of total and active ice contained microbiota from two European ice caves and two South American glacier caves. The impact of anthropogenic polluting activities on the composition and metabolism of ice caves microbiome, as a model for studying current environmental changes in glacial habitats as a result of human impact will also be analyzed. Novel isolated strains indigenous to the ice from Norway, Romania, Chile and Argentina will be screened for metal nanoparticle synthesis of broad bionanotechnological applicative potential, their ability to produce and excrete secondary metabolites, which may have a potential in cancer medicine by producing inhibitory substances for cancer cell lines and their ability to produce antibiotics. Preliminary results indicate that we have isolated new previously undescribed bacteria that have a great potential for producing anticancer agents. Our project represents a unique comparative biogeography in ice environments, and brings together a transnational multidisciplinary research team. These data will be important to reveal the first identities and functions of the key players in these unique and vulnerable ecosystems due to climate change.

The outcome of the project was to characterize the diversity and metabolism of total and active microcosm from cave ice, and evaluate past, present and future consequences of climate driven ice retreat and human impact on biogeochemical and ecological processes. This was done by studying temporal and spatial changes in ecosystems present in ice and glacier caves from various geographical locations, and to establish a strategic framework for multidisciplinary research in ice ecosystems, including investigation of the applicative potential of stage ice microcosm in nanotechnology development. Specific tasks of the project were i) characterization of the functional diversity and activity of a caveice microbiom using metagenomic and high throughput Sequencing, ii) assessment of the impact of climate dynamics and anthropogenic pollution on microbial communities preserved in this particular glacier environment, and iii) isolation of novel organisms and identify cold adapted nanoparticles.

Glacial environments including Polar regions, frozen lakes, mountain glaciers, and upper atmosphere, are currently the focus of intense research due to their recognized vulnerability to climate change and the increased interest in psychrophilic microorganisms due to their great biotechnological potential. However, there is only limited information available on ice microbiota from ice caves. These highly preserved and relatively unexposed ice environments constitute archives for studying the impact of climate and anthropogenic pollution on the diversity of the ice embedded microbial communities as well as on their metabolic activity. Our proposal represents the first investigation of metabolic diversity and activity of microbial communities from a cave ice environment, to reveal the identities and functions of the key players in this ecosystem, using metagenomic and metatranscriptomic analyses. The project emphasis on a comparative study of total and active microbial communities from ice caves of different climate and pollution exposures, in order to understand the microbial diversity and metabolism in this preserved environment, and to establish a chronosequence model reflecting the impact of both climate and environmental pollution on cave ice microcosm. The selected ecosystems represent perennial ice blocks harbored in limestone caves located in Romania (Scarisoara Ice Cave), and Norway (Svarthamar Ice Cave), and in glacier caves located in Argentina (Viedma Glacier cave) and Chile (Mylodon cave, MY). The results of this proposal will contribute not only to unraveling the microbial activity of an unstudied type of habitat, but also to investigate the effect of climate variations and environment pollution on such natural monuments leading to better management of the sites and to their more efficient protection and conservation.