PN-II-PCE ,

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1 Summarizing scientific report on the implementation of the project entitled "Diversity of archaea inhabiting saline lakes from Transylvanian Basin (Central Romania)", Project code PN-II-PCE , Contract No. 186/2011, during October 2011 October 2013 From October 2011 to October 2013 our project activities aimed at achieving the following objectives: 1) Characterization of the vertical distribution of archaeal diversity along the salinity and temperature gradients of the targeted saline lakes. 2) Evaluation of temporal (seasonal) dynamics of archaeal diversity in sampled saline lakes. 3) Estimation of the impact of human activity on spatio-temporal diversity of prokaryotes belonging to Archaea in investigated salt lakes. Phase/Stage 1. During 2011 (November 2011 February 2012) our activities centered only on field trips aiming at in situ monitoring of physical and chemical parameters during the Winter season in the following lakes: Ocnei, Rotund and Tarzan (Turda, Cluj County); Brâncoveanu, Fără Fund and Mâţelor (Ocna Sibiului, Sibiu County); Ursu and Negru (Sovata, Mureş County). Phase/Stage 2. During 2012, the in situ monitoring of environmental parameters has been undertaken in the targeted lakes paralelled by water and sediment samplings for biological analysis during all seasons. Additionally, aided by a commercial environmental analysis laboratory we performed chemical analyses of nutrients (nitrites, nitrates, ammonium, phosphates, sulfides, etc.) as well as of mineral ions (chlorides, sodium, potassium, magnesium, etc.) in the sampled water layers. Chemical analyses were performed on different water layers and in different seasons for evidence of seasonal and vertical variations. In most investigated lakes (except Lake Brancoveanu) a strong physical and chemical stratification of the water was observed, resulting in the categorization of these lakes as meromictic. Meromictic lakes are generally comprising a top layer (from the surface to about 2 m deep) called epilimnion with obvious seasonal variations in temperature and dissolved oxygen, being moderately saline (3-9%). An intermediate layer of water (between 2.5 and 4 m deep) called chemocline follows, with an abrupt change of salinity (halocline, an increase from 9-10% to 25-30% salinity) and dissolved oxygen (oxicline, from 3-4% to less than 0.2%). Under this transition layer there is a layer of water with quasiconstant physical (temperature, around 14 o C throughout the year) and chemical (salinity %; dissolved oxygen - <0.2% etc.) parameters. This layer is hypersaline and microoxic / suboxic and is termed monimolimnion. The bottom layer might be the habitat of extremely halophilic prokaryotes. Among these, we identified the presence of archaea both by molecular analysis of total genomic DNA extracted from water and sediment samples and by culture-dependent approaches (the isolates belonging to Halobacterium spp., Halorubrum spp., Halovenus spp., 1

2 Natrinema spp.). We have demonstrated and reported these findings by two papers submitted for publication (briefly described below in the 2013 scientific report). In general we observed that the chemocline acts as a filter or ecological barrier separating two archaeal communities somewhat different in their structure and composition. This observation applied on two disparate meromictic salt lakes: Brancovan and Ocnei. As managerial objectives we aimed at the publication and participation at international scientific meeting. Both objectives were achieved by publication of an article in a peer-reviewed journal (Andrei și colab., 2012, FEMS Microbiol Lett), and by participating with a poster at Extremophiles 2012 International Conference in Seville, Spain. Phase/Stage 3. During January present, but also during 2012 we succeeded in isolating more than 200 strains of archaea in all sampled lakes. The highest success rate (the highest number of isolates) was obtained with the selective medium, low in nutrients, R2A (17-25% total salinity) in Ocnei and Brâncoveanu lakes. Most of the isolates belonged to the genera Haloferax and Halorubrum by showing a high level of genetic similarity (based on 16S rrna gene sequence). A few isolates showed 16S rrna gene similarity below 97%, the threshold at which isolates can be considered as a new species. During 2013 we tested the morphological, physiological, biochemical and growth features of several isolated with potential to be described as a new species, and the investigation is still ongoing. Worth mentioning our efforts to clarify the affiliation of all isolates by molecular approach (by sequencing performed by an accredited company), activities that took place almost continuously since May of 2012 until now. Following the last field trips (October 2013), in our laboratory we have obtained other isolates whose identification is planned in the coming weeks. Also in this last period of Stage 3, within the main objective of multiannual surveying of physico-chemical and biological factors, we tried to keep a steady rhythm of monitoring the studied salt lakes. The impact of human activity can be investigated only by comparing changes in the medium term (2-3 years) of the biotic and abiotic factors in lakes exploited for recreational purposes (Ocnei, Rotund, Ursu) or directly affected by domestic pollution (Matelor Lake), and in strictly protected lakes (Fara Fund Lake reserve at Ocna Sibiului). Related activities and data processing are under way. In conclusion, the results obtained so far are very good, resulting in the ISI paper (see Andrew et al., 2012), a chapter in a book published at Springer (Banciu and Sorokin, 2013), and by two manuscripts submitted to journals with impact factor over 2.0. Also, we had participated in an international conference, we had an invited presentation, two abstracts at national conference and an article published in an IDB-indexed Romanian journal. Note that reducing the budget for 2013 resulted in rethinking and re-planning of research, and has affected our original intention to have a participation in international conferences. We opted for a strategy of investment in laboratory activities intended to provide publishable results with international visibility. 2

3 Scientific perspectives and potential of social involvement In the future, our activities will bring more evidences on: 1) the assumption that two adjacent extreme habitats with similar environmental characteristics would be populated by similar communities. Our first observations on Ocnei and Rotund Lakes foresee the surprising conclusions; 2) the composition of microbial populations that inhabit the sediment and water layers of lakes Ursu and Fara Fund that need detailed (meta)genomic studies; 3) the impact of human activity on the structure and composition of haloarchaeal communities in exploited and unaffected lakes. All these approaches are possible with predictable and continued funding of this project, the results with a high potential to have a greater national and international visibility. We intend, however, that at the end of the project, expected in 2014, to initiate a public debate supported by a popular action and public awareness on environmental uniqueness and relevance of salt lakes in the Transylvanian Basin. We emphasize that the Project Manager, Horia Banciu, is a permanent collaborator of scientific magazine (Terra Magazin, ISSN , and collaborator of Radio Romania Cultural, being invited for several times in Pro Natura and Știința în Cuvinte Potrivite broadcasts ( In this way, the project manager has a direct access to the mass-media communication facilities. Progress Scientific Report during January October 2013 Background During 2013 we aimed at two major scientific objectives whose accomplishment enabled us writing two articles submitted for publication in journals with international visibility: Extremophiles, and Applied and Environmental Microbiology, respectively. Besides the aims described below, we continued the planned activities of in situ monitoring of physico-chemical parameters of water and sediment sampling in Rotund (Cluj) Fara Fund (Sibiu) and Ursu lakes (Mureş). Additionally, we isolated archaeal strains. Total genomic DNA from environmental samples allowed the characterization of prokaryotic molecular diversity. In 2013, the two main objectives were: I. Assessment of multiannual and seasonal dynamics, and spatial distribution of archaeal diversity in hypersaline, meromictic Ocnei Lake (Turda region, Cluj County) and II. Investigation of effects led by environmental factors on the vertical distribution and archaeal community structure in the hypersaline Brâncoveanu Lake (Ocna Sibiului, Sibiu County). 3

4 Objective I. Assessment of multiannual and seasonal dynamics, and spatial distribution of archaeal diversity in hypersaline, meromictic Ocnei Lake (Turda region, Cluj County) Introduction Ocnei ( Salt mine s ) Lake lies on the western border of the Transylvanian Basin, owing its formation to the collapse and flooding of an old salt mine from the Middle Ages (Alexe, 2010). It is one of the most important and representative lacustrine unit from the existing nine lakes in Turda area (Cluj County), mainly because of its considerable depth (ca 33 m) and volume (ca 26,000 m 3 by 2001) (Alexe & Furtună. 2012). Archaeal diversity has been described in numerous hypersaline ecosystems (salt lakes, soda lakes, springs, solar salterns, ancient salt deposits) and although these habitats are considered extreme they harbor diverse microbial communities (Benlloch et al., 2002; Gruber et al., 2004; Baati et al., 2010; Youssef et al., 2012). Few studies on the structure and diversity of archaeal communities inhabiting meromictic saline and hypersaline lakes are currently available and none focused on spatial and/or temporal distribution of the archaeal population (Bowman et al., 2000; La Cono et al., 2013). The aim of present study was to get insight on the seasonal dynamics of archaeal diversity distributed along the physical and chemical gradients in the water column of the hypersaline, meromictic lake Ocnei. Materials and methods Sampling site and sampling methodology. Ocnei Lake (46 o 35 N and 23 o 47 E) is located at an elevation of 360 m, in Turda area (Cluj County), and it lies on the western border of the Transylvanian Basin, in a diapiric area, that appears in the open under the form of salt massifs. These kinds of structures have been exploited even since the Roman period. This lake has been developed on an old salt mine from the Middle Ages ( bell system mine) as a result of natural accumulation of waters in the abandoned mines or in the cuts that have remained after the collapse of galleries. Ocnei Lake has a circular shape, characteristic to bell types of salt lakes, with a small surface area (round 2200 m 2 ), steep morphological profile, considerable depth (33.3 m) and a total volume of 26,250 m 3 (Alexe & Furtună. 2012). Ocnei Lake is meromictic, with saline epilimnion and hypersaline chemocline and monimolimnion. During late summer, the upper water layer (1.5-3 m depth) often reaches temperatures of over 30o C, in a phenomenon called heliothermy. Sampling was performed at approximate middle of the lake, seasonally during The temperature, salinity, dissolved oxygen concentration and ph of the water were measured in situ with a portable water multiparameter (HI 9828/20, Hanna Instruments, USA). Culture media and growth conditions Members of the Halobacteriaceae family were isolated under aerobic conditions on different growth media. Medium I consisted of (g L -1 ): NaCl 120, 175, 220, MgSO4.7H2O 20, KCl 5, peptone 5, yeast extract 5, CaCl2.6H2O 2, agar 20, ph ~ 7.2 (Atlas, 2004). R2A medium consisted of (g L -1 ): NaCl, 120, 175, 220 and agar R2A, 20, ph 7.2. Ocnei Lake medium consisted of filtered lake water to which peptone (5 g L-1) and yeast extract (2 g L -1 ) and agar (20 g L -1 ) were added before sterilization. Different plating approaches were undertaken: serial dilutions of lake water, direct plating of 100 µl of sample water, spread on the plate or filtration 4

5 of 1 ml of sample onto 0.2 µm-pore-size mixed cellulose ester membrane filters which were placed on the plate. All culture plates were incubated aerobically at 35oC in sealed plastic containers for at least 4 weeks. Selected colonies were analyzed by PCR amplification of 16S rrna gene and sequencing as described below. DNA extraction and amplification of 16S rrna genes Water samples (100 ml) were filtered through sterile 0.2 µm-pore-size mixed cellulose ester membrane filters (diameter 45 mm) to collect microbial biomass. After filtration the membrane filters were stored at -20oC until DNA extraction. Genomic DNA from environmental samples was extracted using ZR Soil Microbe DNA kit (ZymoResearch, USA) according to the manufacturer s protocol. Genomic DNA from haloarchaeal pure cultures was extracted using GeneJet Genomic DNA Purification kit (Thermo Scientific), according to the manufacturer s recommendations. 16S RNA genes from isolated haloarchaeal strains and environmental samples were amplified by using archaeal specific forward primer 5 -TCYGGTTGATCCYGCCRG-3 in combination with the universal reverse primer 5 -GGTTACCTTGTTACGACTT-3. The PCR was performed with Bio-Rad T100 TM Thermal Cycler (Bio-Rad, USA) using the following conditions: 94 o C for 2 min, followed by 30 cycles of 94 o C for 45 s, 53 o C for 1 min and 72 o C for 1 min and 20 s, with final 10 min extension at 72 o C. Sequencing and sequence analysis The clones were partially sequenced by Sanger method by a commercial company (Macrogen Europe, The Netherlands). Approximately 1100 partial 16S rrna gene sequences were grouped into operational taxonomic units (OTUs) using the default parameters in the QIIME package. The output files created by QIIME were further used to construct rarefaction curves using FastGroupII online package. Biodiversity within each bacterial clone library was also estimated via the Shannon Weaver index and the Chao1 index. Accession numbers of nucleotide sequences Partial 16S rrna sequences obtained from archaeal clones (and strains) used for phylogenetic analyses as described above have been deposited in the NCBI database under accession numbers KF KF (archaeal clones) and KF KF (archaeal strains). Results Spatial and temporal dynamics of environmental parameters As far as salinity is concerned, Ocnei Lake is hypersaline, the salt concentration reaching 322 g L -1 in the deeper layers. Above the chemocline, salinity values varied between g L - 1, due probably to surface freshwater input as rainwater alternating with intense evaporation periods. Salinity showed weak seasonal fluctuations, visible in the epilimnion, generally being higher in summer/early autumn and lower in winter and spring (Figure 1A). Salt concentration gradually increased with depth with a steep halocline between 3 and 4 m, reaching constant values below 5 m. To get insight on the main chemical contributors to the total salinity, we analyzed the relevant cations and anions in the water sample collected during spring season at 3.5 m depth. We found that Na + (91.4 g L -1 ), Mg 2+ (0.397 g L -1 ), and K + (0.078 g L -1 ) were the 5

6 main cations, while Cl - (112 g L -1 ), SO 4 2- (1.13 g L -1 ), and HCO 3 - (0.376 g L -1 ) were the most important anions present in water. In this regard, the ionic composition of Ocnei Lake is similar to that of the seawater. ph parameter showed same trend, changing from slightly alkaline ( ) in the epilimnion to slightly acidic ph ( ) in the monimolimnion (Table 1). Seasonal variations in temperature were recorded in the upper 4 m of the water column, with significant differences between winter and summer. Below 4 m the temperature is constantly at around 17 o C (Figure 1B). The thermocline was generally present between 3 and 5 m. The thermal stratification due to heliothermic phenomenon was obvious during April and November surveys, when temperatures of the intermediate strata (2 m and 4 m, respectively) were higher (18.2 and 24.2 o C) than that of the surface layer (12.5 and 19.5 o C). In July, due to intensive bathing, the epilimnion and chemocline (0.5-4 m) were isothermal (26 o C). Figure 1. Depth profiles and seasonal variations of salinity (A), temperature (B), dissolved oxygen (C) and total cell counts (D) in water column of Ocnei Lake. Symbols: diamonds - January; squares - April; circles - July; triangles - November. Dissolved oxygen (DO) concentration showed strong seasonal variations in the epilimnion fluctuating between a maximum of 7.23 mg L -1 at surface (April 2012) and a minimum of

7 mg L -1 at same sampling point (November 2012) (Figure 1C). However, during all seasons, a second peak of DO parameter was noticed at around 4 m. Concentration of DO decreased with depth and salinity, sharply reaching values close to zero ( mg L -1 ) just below the chemocline (5 m), in the upper part of the monimolimnion. Isolation of haloarchaeal strains A total of 43 archaeal isolates were obtained during the four sampling periods. All isolates were related to known haloarchaeal phylotypes described in other similar hypersaline environments and formed 10 OTUs (Table 1). Archaea belonging to Halobacteriaceae family have grown on all media used in this study but the highest numbers of archaeal isolates (27) have been achieved by membrane plating on R2A agar medium supplemented with 17.5 or 22% NaCl. The most abundant group was represented by the Halobacterium strains (41.8% of all isolates) followed by Haloferax spp. (25.5 %) and Halorubrum spp. (20.9%). Species belonging to these three genera were also identified in the clone libraries constructed. Although we were not able to grow the representatives of the group most frequently retrieved in the clone libraries (Halogeometricum rufum related sequences), Halobacterium and Halorubrum were next most abundant phylotypes detected by molecular methods. Table 1. 16S rrna gene-based identification of archaeal isolates from water column of Ocnei Lake. Closest NCBI match, with accession number and percentage of sequence identity are given. Strain No. of isolates Closest GenBank sequence match Taxon Accession No. 7 % Identity 1 16 Halobacterium salinarum R1 NR_ Halobacterium jilantaiense AB Haloferax volcanii E8 HVU Haloferax prahovense RS80 KC Haloferax prahovense TL6 NR_ S 6 2 Halomicrobium mukohataei EF Halorubrum saccharovorum NR_ Haloferax alexandrinus JX Haloarchaeobius iranensis AB Halopenitus persicus strain DC30 JF Archaeal diversity estimated by clone libraries. The sequences of 1152 clones have been clustered within 40 OTUs, the majority being closely affiliatedto members of Halobacteriaceae (Figure 2). Four clones were found to appertain at Methanocaldococcaceae, and one OTU was found with 93% similarity with 16S rrna gene sequence of Candidatus Nitrososphaera (Thaumarchaeota). Statistical analysis of sequences and environmental variables indicated an

8 increase of diversity in a salt-dependent manner. The most obvious difference was observed between the top water layer (epilimnion) acommodating 8 different genera and a dominant phylotype, and the bottom, suboxic layer (monimolimnion) populated by 16 different genera and several dominant phylotypes. Figure 2. Abundance of haloarchaeal OTUs at different depths of the water column in Ocnei Lake. The bar chart compares the diversity of 16S rrna gene sequence of OTUs recovered from all seasons (1O - winter, 2O - spring, 3O - summer, 4O - autumn) and depths (0.5 m - epilimnion, 3.5 m - chemocline, 11 m - monimolimnion). Minor members include genera with abundances below 2%.. Concluzii 1. The results indicated clear differences between Archaea communities present in the upper layer, well-oxygenated and subject to seasonal variation of physical-chemical parameters and the thermally and chemically stable deep layer; Biodiversity was found to increase with depth and salinity, most numerous archaeal phylotypes being present in the microoxic/suboxic hypersaline layer (monimolimnion). 2. Seasonal variations of physico-chemical parameters influenced the distribution and diversity of archaeal communities in the epilimnion only. 3. Archaea communities in Ocnei Lake is dominated by species belonging to the family Halobacteriaceae, including common genera found in other in hypersaline environments (Halobacterium spp., Halorubrum spp., Haloferax spp.) but also recently characterized genera such as Halopenitus and Haloarchaeobius. 8

9 II. Investigation of effects led by environmental factors on the vertical distribution and archaeal community structure in the hypersaline Brâncoveanu Lake (Ocna Sibiului, Sibiu County). Introduction Unveiling the mechanisms underlying the spatial structuring of microbial communities and the manner by which the assemblages respond to environmental variations are major challenges in microbial ecology. Even though studies have shown that environmental filtering is one of the main mechanisms of community assembly in prokaryotes, it was revealed that in some cases the dynamics of microbial community assembly follow predictions made by the neutral theory. Thus, even if the naturally occurring communities of prokaryotes are vital to the biogeochemical cycles and catalyze processes that are critical to sustaining life on Earth, the rules that govern the foundation of their communities are barely understood. The study of microbial associations in hypersaline habitats is well established and although salt-adapted organisms derive from all three domains of life, most hypersaline ecosystems are dominated by halophilic archaea. These habitats characterized by low biodiversity have proven useful as model systems in corroborating molecular (eco-)systems biology approaches and application of ecological and evolutionary theory. Furthermore, they serve as valued models in establishing linkages between community structure and function, in uncovering the processes that determine the spatial structure of microbial assemblages, and the mechanisms by which they respond to environmental changes. The community ecology of hypersaline environments has its fundament on experiments performed on saline salterns and the majority of studies that investigate the influence of environmental factors on microbial diversity were focused on temporally dynamic systems. The athalassic meromictic hypersaline lakes, due to their unique limnological features and physicochemical characteristics (e.g. constant vertical stratification, steep chemical gradients, oxygen deprived zones), have the potential of providing new insights regarding the ecology of microorganisms that thrive therein. Taking into consideration the aforementioned rationales, the present study primarily focused on exploring the extent of niche-based ecological interactions (i.e. environmental filtering, competition) in structuring the archaeal diversity, assessing the predictive capacity of environmental factors over the spatial distribution of archaeal assemblages, and in estimating the environmental impact of archaea by determining its degree of dominance over the bacterial counterpart. Summarized Results: 1. Statistical analysis of physico-chemical parameters in Brancoveanu Lake using "cluster" and ANOSIM showed that they form two distinct groups: one at surface and one at deep, predicting the existence of at least two distinct communities of Archaea. Molecular fingerprinting analyzes (ARDRA and DGGE techniques) revealed the presence of two types of communities throughout the water column in accordance with the distribution of the physico-chemical parameters. 2. Quantification of the number of prokaryotes belonging to Archaea and Bacteria was performed using Real-Time qpcr technique. The results indicated that the number of archaea range from to cells/ ml, and the number of bacteria varied between and cells/ ml. Statistical results of analyzes showed that the 9

10 number of archaea is directly related to ph, oxidation-reduction potential and salinity, unlike bacteria which is related to temperature. 3. Cultivation of microorganisms on selective media allowed identification of strains, some being previously uncharacterized and with potential for applicative research. 4. Analysis of archaeal biodiversity performed by construction of clone libraries, the 16S gene sequencing and bioinformatics analysis. The results showed that almost all sequences retrieved belonged to Halobacteriaceae family, and that the two depth-related communities are slightly different in terms of their species composition (Fig. 3). 5. Estimation of the impact of human activity on microbial biodiversity was assessed by seasonal sampling. The results obtained using ARDRA and DGGE techniques have shown that archaea communities are not influenced by human activities carried out during the analyzed seasons. 6. Canonical correlation analysis (CCA) showed that co-ocurrence and deterministic processes contribute to the assembly of archaeal communities, and that at local scale, the salinity affected them most. Figura 3. Heat map depicting archaeal diversity and the relative abundances of each OTU (defined at a 97% sequence identity cutoff) in water samples collected from Brâncoveanu Lake at two different depths (-0.5 m and -7 m). Conclusions 1. The deterministic mechanisms (selective pressure of environmental factors) were found to shape the assembly of archaeal communities in Brancoveanu Lake. 2. Brâncoveanu Lake is apparently inhabited by novel lineages with unexplored ecological and applicative capabilities. 10

11 3. Apparently, the human activity did not affect the species diversity belonging to Archaea in Brancoveanu Lake. References Alexe M & Furtună P (2012) Natural and anthropic risks in the area of Durgău-Valea Sărată Turda salt lakes. Water resources and wetlands (Gâştescu P, Lewis W Jr. & Breţcan P, eds), pp Tulcea, Romania. Benlloch S, López-López A, Casamayor EO, Øvreås L, Goddard V, Daae FL, Smerdon G, Massana R, Joint I, Thingstad F, Pedrós-Alió C & Rodríguez-Valera F (2002) Prokaryotic genetic diversity throughout the salinity gradient of a coastal solar saltern. Environ Microbiol 4: Falkowski PG, Fenchel T, Delong EF The Microbial Engines That Drive Earth s Biogeochemical Cycles. Science 320(5879): Ghai R, Pašić L, Fernández AB, Martin-Cuadrado A-B, Mizuno CM, McMahon KD, Papke RT, Stepanauskas R, Rodriguez-Brito B, Rohwer F, Sánchez-Porro C, Ventosa A, Rodríguez- Valera F New Abundant Microbial Groups in Aquatic Hypersaline Environments. Sci. Rep. 1:135. doi: /srep Gruber C, Legat A, Pfaffenhuemer M, Radax C, Weidler G, Busse HJ & Stan-Lotter H (2004) Halobacterium noricense sp. nov., an archaeal isolate from a bore core of an alpine Permian salt deposit, classification of Halobacterium sp. NRC-1 as a strain of H. salinarum and emended description of H. salinarum. Extremophiles 8: Jones SE, McMahon KD Species-sorting may explain an apparent minimal effect of immigration on freshwater bacterial community dynamics. Environ. Microbiol. 11: Lauro FM, DeMaere MZ, Yau S, Brown MV, Ng C, Wilkins D, Raftery MJ, Gibson JA, Andrews-Pfannkoch C, Lewis M, Hoffman JM, Thomas T & Cavicchioli R (2011) An integrative study of a meromictic lake ecosystem in Antarctica. ISME J 5: La Cono V, La Spada G, Arcadi E, Placenti F, Smedile F, Ruggeri G, Michaud L, Raffa C, De Domenico E, Sprovieri M, Mazzola S, Genovese L, Giuliano L, Slepak VZ & Yakimov MM (2013) Partaking of Archaea to biogeochemical cycling in oxygen-deficient zones of meromictic saline Lake Faro (Messina, Italy). Environ Microbiol 15: Woodcock S, Van Der Gast CJ, Bell T, Lunn M, Curtis TP, Head IM, Sloan WT Neutral assembly of bacterial communities. FEMS Microbiol. Ecol. 62: I hereby confirm the corectedness of the above information 18 October 2013, Cluj-Napoca Project Manager Assoc. Prof. Dr. Horia BANCIU 11