The methodology proposed for implementing these objectives entails the following activities and techniques, indicating the implementation period (years) throughout the project. Considering the terminal year 2011 of PCE project ID_1034, the activities of this project for 2011 are limited to documentation and partial inventory acquisition.

Objective 1.
Phylogenetic and physiological study of the microbial diversity
in ice deposits from Scarisoara Cave using culture-dependent and culture-independent approaches

Activity 1.1 Ice sampling from diverse locations in Scarisoara Cave (Years: 2012, 2013)

Ice samples will be collected from ice layers of different ages and geochemical composition from the perennial ice block of the Little Reserve, and from the ice lake of the Great Hall from Scarisoara Cave. Two sampling sessions are planned during this project, in 2012 and 2013, respectively. The second sampling session is scheduled in accordance with advancement of undergoing dating studies of the ice block. Ice samples will be extracted from the layers of the ice wall of Little Reserve at 100 years time interval, from fractions containing high and low concentration of organic matter. Ice cores will be extracted under sterile conditions, using specific drilling equipment, and stored at -80°C until further used. Water samples from the ice lake of the Great Hall will be collected and filtered on sterile 0.2 mm membranes.

Activity 1.2 Cultivation of microorganisms from collected ice samples (Years: 2012, 2013, 2014)

Cultivation of microorganisms from collected ice samples will be carried out on various media (rich, mineral, selective) and under specific conditions (temperature, pH, salinity, light) for isolating microbiota and different physiological groups of microorganisms (Fe-reducers/oxidizers, S-reducers/oxidizers, N-fixing prokaryotes, methanotrophs, cyanobacteria, etc.) from various locations of Scarisoara’s ice block. Liquid and solid media will be inoculated with ice samples and incubated at different temperatures and time periods. The resulted liquid cultures will be used for extraction of total DNA and SSU rRNA gene library construction. Isolated colonies will be purified and submitted to microbiological and biochemical investigations for future identification of strains of applicative value.

Activity 1.3 Microbiological characterization by epifluorescence microscopy and flow cytometry (Years: 2012, 2013, 2014)

Epifluorescence microscopy and flow cytometry analyses will be performed on ice samples and cultured enriched populations, to reveal the presence and content of eukaryotic and prokaryotic microorganisms, including ultrasmall cells, and measure the number of total and live cells, the ability to grow and multiply (Kogure method), cell dimensions, DNA content, dry cell biomass in ice samples and cultures obtained on selective media, and their physiological state at single cell level (intact plasma membrane, intracellular esterase and dehydrogenase activities) (Williams et al., 1998; Joux and Lebaron, 2000). Acquisition of top-rank equipment for performing both these techniques is in process, and a LIVE/DEAD cell viability kit will be used for ice sample staining.

Activity 1.4 Assessment of phototrophic carbon and nitrogen fixation (Years: 2012, 2013, 2014)

Autotrophic cyanobacteria, growing on inorganic substrate, atmospheric CO2 and N2, play an important role in sustaining autotrophic communities. Therefore, the role of cold-adapted cyanobacteria in the C- and N-cycles will be studied in ice/water interface from light exposed lake from Great Hall of Scarisoara Cave. Phototrophic carbon fixation and nitrogen fixation in light and dark by microbiota from ice samples or enriched microbial cultures will be monitored using photosynthetic oxygen measurement with oxygen electrode and gas chromatography, respectively.

Activity 1.5 Construction of SSU rRNA gene libraries (Years: 2012, 2013)

Small subunit (SSU) rRNA gene libraries for prokaryotic 16S rRNA and eukaryotic 18S rRNA genes will be obtained from ice samples collected from layers corresponding to different ages and geochemical compositions, as well as cultures obtained under different growth conditions:

  1. Extraction of genomic DNA from ice samples and microbial cultures Total DNA from ice samples will be extracted using water and soil genomic DNA extraction kits (MoBio), and from microbial cultures using a tissue genomic DNA extraction kit (Qiagen). In both cases, an additional lysis step will be performed in the presence of mutanolysin (Sigma).
  2. PCR amplification of SSU-rRNA genes SSU rRNA genes specific for each microorganism’ domain (phylum) Bacteria (cyanobacteria), Archaea, eukaryotic microorganisms (protists, fungi, green algae) and microscopic arthropods will be amplified by PCR using corresponding universal and specific primers.
  3. Clone library construction The amplified SSU rRNA DNA fragments will be inserted in pGEM-T Easy cloning vector (Promega) and the resulted plasmid library will be used for transformation of E. coli XL1Blue. The 16S/18S-rRNA gene libraries obtained will be stored as bacterial cultures at -80°C.

Activity 1.6 Diversity screening of gene libraries by DGGE and ARDRA (Years: 2012, 2013)

The molecular diversity of the SSU rRNA gene libraries obtained will be analyzed using Denaturing Gradient Gel Electrophoresis (DGGE) and Amplified Ribosomal DNA Restriction Analysis (ARDRA) by PCR gene amplification and restriction enzyme digestion (Muyzer et al, 1993; Vaneechoutte et al., 1998). The plasmids from each group with similar fragment patterns will be purified using plasmid miniprep kits. Their concentration will be measured using Nanodrop1000.

Activity 1.7 Sequence determination of SSU rRNA gene libraries (Years: 2012, 2013)

All purified plasmids presenting variations from previous screening steps will be sequenced (both nucleotide strands) using a Genetic Analyzer ABIPrism 3500 (Applied Biosystem). The gene sequence will be examined using Blast Software from NCBI web site for species identification. Nucleotide sequences of the 16S and 18S rRNA gene clones and isolates used in the phylogenetic analysis will be introduced in GenBank database.

Activity 1.8 Phylogenetic analysis (Years: 2013, 2014)

Based on DNA sequences of 16S/18S rRNA gene libraries obtained from different ice samples and cultures from the same ice location, phylogenetic trees will be generated by multiple alignments and distance analysis using neighbour-joining algorithm of Juke-Cantor model. The resulted phylogenetic trees illustrating the biodiversity of microorganisms of each ice sediment, will be analyzed in correlation with their age, chemical composition and embedded climate patterns.

Objective 2.
Comparative analysis of microbial diversity in Scarisoara Cave ice sediments
in relation to their age, light exposure, geochemical properties and climate patterns

Activity 2.1 Geochemical analysis of the investigated ice samples (Years: 2012, 2013, 2014)

Physico chemical parameters (temperature, pH) of all ice samples will be measured on site during sampling sessions. The chemical composition of prelevated ice samples will be analyzed in respect to total organic carbon and nitrogen content by TOC analysis, and to cathion and anion composition by atomic absorption spectrometry and x-ray spectrometry.

Activity 2.2 Statistical analysis of microbial diversity in different ice locations (Years: 2013, 2014)

The species’ composition from each ice location will be analyzed in relation to the age and chemical composition of the ice substrate, and to the growth temperature, to determine specific community patterns. Statistical analysis based on biodiversity Shannon indices (Hill et al., 2003) will be performed for calculating the distribution of major phylogenetic groups in ice samples.

Activity 2.3 Quantitative analysis of microbial diversity by qPCR (Years: 2013, 2014)

Based on this qualitative analysis, the quantitative microbial distribution of selected species across the ice layers from the ice block of Little Reserve in Scarisoara Cave will be determined in order to study possible differences in the microbial abundance from different aged-ice layers. The abundance of microorganisms belonging to different physiological groups (Fe-reducers/oxidizers, S-reducers/oxidizers, N-fixing prokaryotes, methanotrophs), and of photosynthetic microorganisms (cyanobacteria, etc) from light-exposed ice lakes but also deeper ice layers, will be determined by qPCR amplification of specific genes for each group using RT-PCR thermocycler.

Activity 2.4 Chronological biodistribution analysis and climate biomarkers identification (Years: 2014)

The presence and representation of different species of prokaryotic and eukaryotic microorganisms’ along ice sections of different ages will be cross-analyzed using the qualitative and quantitative results in relation with the chemical composition of ice samples and previously reported geochemical information and climate patterns of Scarisoara Cave, in view of defining specific ice embedded biomarkers for climate events that occurred during the last 1000 years.

Objective 3.
Identification of new psychrophilic and/or psychrotolerant strains
for selecting novel microorganisms of biotechnological interest

Activity 3.1 Isolation and characterization of newly identified strains (Years: 2013, 2014)

Based on the identified 16S/18S rRNA gene sequences from the constructed gene libraries (activity 1.6), the isolation of new strains (bacteria, archaea, and/or eukaryotic microorganisms) will be performed on selective media under different conditions (temperature, pH, light intensity) that correspond to their closest species’ growth conditions, and confirmed by sequencing of respective SSU rRNA gene amplified from the extracted genomic DNA.

The morphological and physiological properties of newly isolated strains will be performed using standard microbiological methods, electron microscopy, and biochemical tests.