Almost half of Slovenian territory (44%) is composed of limestone, which is highly permeable and soluble sedimentary rock. Limestone dissolution has created surface and underground karst topology with more than 10000 registered caves (10871 caves on 9 January 2014, Cave cadastre of the Karst Research Institute ZRC SAZU) and large groundwater reservoirs. Groundwater reservoirs are important from many points of view. First, these reservoirs provide freshwater supply around half of the Slovenian population (Ravbar, 2007). Second, some of these habitats are still pristine. This is important because human activity and pollution changes the composition of microbes in natural populations and dramatically decreases its diversity. Third, groundwater reservoirs are susceptible to pollution and represent important sites for gaining better understanding of how humans interact with fragile environmental systems. 

The quality of karst groundwater in Slovenia and elsewhere and the putative presence of pathogens are not properly monitored (Culver et al., 2012). Accordingly, the diversity of microbes that are native to groundwater reservoirs and their interaction with human introduced ones remains virtually unknown compared to their counterparts in other aquatic systems. A GenBank search in March 2014 returned approximately 9×104 hits from groundwater studies compared to 5×105 hits from freshwater and 5×106 hits from studies conducted in marine environments. And yet, this overlooked habitat is an exciting system to study from the point of view of a microbial ecologist. It is assumed that the groundwater in karst supports high prokaryotic diversity as it contains mixtures of aquatic and terrestrial bacteria introduced through percolating water and activer or intermittent river flow. In addition, groundwater likely hosts metabolic specialists that are able to degrade complex compounds present in water. Groundwater in Karst is also expected to host ultramicrobacteria, microbial cells that are less than < 0.1 μm3 in size and whose presence has been noted in other freshwater systems (Hahn et al.,2003) and have been completely overlooked in karst studies.In the past ten years, the advances in sequencing techniques have permitted obtaining large datasets of environmental DNA sequences without the need for prior cultivation. This novel field of metagenomics has unveiled the extent of genetic potential present in the environment. Metagenomics proved to be a versatile tool in microbiology and extended beyond gene cataloging. Metagenomics alone or in combination with analysis of large insert fosmid libraries or flow cytometry based cell sorting, metagenomics permits insight into genomic diversity of microbial populations (Coleman et al., 2006; Pašić et al., 2009; Gonzaga et al., 2013) and even reconstruction of entire genomes from the environment (e.g. Tyson et al., 2004; Mizuno et al., 2013a). In fact, metagenomic and fosmid libraries proved to be archival repositories that can be studied for many years and that provide material for further, more specialized studies, e.g. searching for certain metabolic pathways. The lack of exploration of microbial biodiversity in Slovenian karst groundwater reservoirs and the rich biodiversity of this area (Culver et al., 2012) make it an environment of special interest for metagenomic studies. Accordingly, we propose to take the advantage of novel metagenomic techniques in order to generate basic knowledge on groundwater microbiota of Slovenian karst in the form of genomic sequence databases.

Specific aims of the project:

Specific aim 1. Identify sites of interest The groundwater bodies of Slovenian karst are monitored in terms of water quality by the Environmental Agency of the Republic of Slovenia. Many underground water connections with hydrodynamic properties are already studied and known and were carried out by the Karst Research Institute ZRC SAZU. We are interested in sampling pristine with noor minimum human impacted groundwater and therefore, the sites of interest will be identified using the data available from these two institutions. Specific aim 2. Generate a metagenomic database using environmental DNA extracted from pristine groundwater. We plan to study groundwater samples with the respect to suspended microbiota and microbes that are attached to particulate material. We find the attached microbes very interesting because they can be classified via genomics into ecotypes based on the size of particulate matter they utilize. This supports the idea that genomic diversity within the microbial population allows broad responses to variances in the environment. We are also interested in metagenomic analysis of ultramicrobacteria, a group of frequently overlooked cells. We further plan to sample groundwater at different times of year in order to establish temporal and seasonal dynamics of microbial population. In this manner, we expect to obtain a comprehensive image of the microbial community living in this essential freshwater habitat and identify taxa of interest for Specific aims 3 and 4.

Specific aim 3. Isolate culturable microorganisms at specific hydrological events to compare with metagenomic findings. Recently, a number of studies reported advances in improving the diversity in cultures of prokaryotes due to inclusion of essential nutrients and/or signaling molecules from the native environment in cultivation media (Vartoukian et al., 2010). We plan to employ these improvements to isolate cultivable microorganisms of interest at specific time points to compare with metagenomic findings and investigate strain differences. Specific aim 4. Obtain genomic information on uncultivable organisms through assembly of metagenomic data and singlecell genomics We plan to use two approaches to obtain genomic information on ecologically and/or taxonomically important, yet uncultivable organisms. The first approach is direct genome assembly from metagenomic data. This approach yields reliable, nonchimeric results only if microorganism of interest is predominant in the environment and if its population is composed of a relatively small number of clonal lineages. The second approach comprises sorting individual microbial cells using fluorescenceactivated cellsorting followed by singlecell genomics. The knowhow and the advice on bioinformatics analysis will be provided by group of prof. Francisco Rodriguez Valera at University Miguel Hernandez, Spain.Based on the data from cultivation and metagenomic analyses we will: (i) assess the total diversity of the selected underground aquifers, which will enable us to compare microbial communities among pristine aquifers and human impacted ones and (ii) establish a database based on metagenomic data which will allow further gene mining for specific genes, e.g. catabolism of pesticides and other xenobiotics.