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-2011-3-0546, Contract No. 186/2011, during October – December 2011 Introduction Transylvanian Basin is an important geographical area acommodating numerous saline pools and lakes formed on salt deposits of marine origin dating from Miocene (13.8 Ma, de Leeuw et al., 2011). Substantial accumulation of salt in the Transylvanian Basin are found in Ocna Dej, Turda, Ocna Mures Ocna Sibiu, Jabenita, Sovata. Transylvanian Basin salt lakes were formed either naturally (e.g. Bear Lake, Alunis Lake, Green Lake, Red Lake) or anthropogenic. The anthropogenic salt lakes appeared at the place of the old salt exploitations during Daco-Roman period (e.g. Ocna Dej, Turda, Cojocna). Most of the Transylvanian Basin salt lakes are stratified (meromictic) lakes presenting heliothermy phenomenon. Variation in the physico-chemcal properties of saline and hypersaline environments properties (mono- and divalent cations and anions, dissolved mineral content, pH) is reflected in the diversity of the communities of microorganisms that live in those conditions. Halophilic and extremely halophilic microorganisms are found in all three domains of life (Archaea, Bacteria and Eukarya) given the high degree of diversity of physiological, biochemical, and genetic capabilities (Oren, 1999). Halophilic archaea are common within Halobacteriaceae, Methanospirillaceae, Methanosarcinaceae famillies; within the domain Bacteria, the halophilic species are distributed in all phyla and orders being extremely varied in terms of physiological properties: aerobic and anaerobic; chemo- and photoheterotrophic as well as chemolithotrophic bacterial members (Ollivier et al., 1994; Oren , 1999); the diversity of eukaryotic microorganisms capable of growing under conditions of high salinity is relatively low: green algae belonging to genus Dunaliella , Picocystis salinarum , and some diatoms that can grow in salt concentrations up to 150 g / l (Roesler et al. , 2002; Lewin et al., 2000, Clavero et al., 2000). Abiotic factors relevant to diversity of prokaryotic and eukaryotic communities of salt lakes are: salinity, redox potential, ion concentration, oxygen concentration, temperature and pH.

1 Specific objectives achieved 1. Measurement of physico-chemical characteristics of water; 2. Estimating primary production of phytoplankton by determining the chlorophyll concentration; 3. Determination of abundance picophytoplankton; 4. Determination of the total number of bacterial cells.

Materials and methods Site description and water sampling. Bottomless Lake (Tarzan Lake or Fara Fund Lake, Turda) (46o34'53 "N, 23o49'29''E, altitude 356 m) has almost circular shape, slightly NE-SW elongated, with a diameter of about 65 m. Below the waterline, lacustrine basin has largely shape of a truncated cone with small base facing down. On November 4, 2011, water samples from different depths and surface were collected in Falcon tubes (50 ml) and bottles (2L) using a peristaltic pump and kept on ice or at -20 ° C until further analysis. Laboratory filtration of water samples were performed using a Millipore glass microfiltration system connected to an electric vacuum pump. Physico-chemical characteristics of the water . Physico-chemical parameters (temperature, salinity, electrical conductivity, pH, redox potential, dissolved oxygen) were determined in situ using a WTW multiparameter. Concentration of chlorophyll a. Water samples were filtered through a glass fiber filter GF / 5, d = 47mm (Millipore). Chlorophyll was extracted with methanol: the filters were placed in glass tubes and incubated with 5 ml of methanol at least 5 minutes after which they were heated to the flame for one minute. After extraction, the samples were centrifuged at 5000 x g for 10 minutes. Chl a concentration was determined spectrophotometrically by measuring the absorbance was methanol extract at 750 nm, 666 nm and 653 nm and using the following formula (A666,653,750 extract absorbance value at the three wavelengths, VA represents the volume of filtered water and VM volume methanol; Keresztes si colab., 2010): Chl-a ( µg/l) = [17.12 * (A666 – A750) – 8.68 * (A653 – A750)] / (VA/VM) * 100] Determination of phytoplankton abundance. To filter the samples (10 ml) MCE black gridded membranes (pore size of 0.45 um, d = 47 mm) were used. Membranes were fixed on microscope slides in 50% glycerol, covered with a slide and examined using epifluorescence microscope, Nikon Optiphot 2 at x1000 magnification. Each field was examined using blue- violet excitation light.

2 Total number of bacterial cells. Samples (10 ml) were fixed with 25% glutaraldehyde (15 min at room temperature), after which they were filtered through MCE membranes (pore size 0.45 um, d = 47mm). After filtration the membranes were stained directly using fluorochrome DAPI (4 ', 6-diamidino-2-phenylindole, 5 mg / ml) by incubation in the dark for 15 minutes. After staining, the membrane filters were fixed in 50% glycerol and examined using epifluorescence microscope OLYMPUS BX 51, using DAPI filter Results In total, ten samples were collected in the Bottomless Lake along a water column with 6 m depth. The values of physico-chemical parameters (Table 1) indicated a thermal and chemical stratification of the lake (Figure 1). The depth of the surface layer is 3 m, the temperature being maintained between 6,6-7.2oC; below 3 m depth the water temperature increased by more than five units, the value recorded at 4 m being 13.1oC. It was also observed an increase in salinity for over 10 units, from 33.6 g/L in surface to 45.1 g/l at 6 m depth. The pH in the surface layer was approx. 8.7, indicating a slight alkalinity of the top layer. The bottom layer (3-5 m depth) showed a pH around neutral. Dissolved oxygen was between 56% - 64.5% in the surface layer, while below 3 m depth, the DO decreased by more than 30 percent. Water transparency was optimal up to 3.7 m. Table 1. Physical and chemical parameters and Chl-a concentration in Bottomless Lake (Turda, Cluj County) during November 2011. Sample Physical and chemical parameters Chl-a no./ Redox concentration o Depth T( C) Salinity Conductivity pH potential O2 O2 µg/l (m) (g/l) (mS/m) (mV) (mg/l) (%) 1 (0) 7 34 53,5 8,75 -102 7,5 64,5 3,73 2 (-0,5) 6,8 33,7 53 8,7 -102 6,56 55,4 1,09 3 (-1) 6,6 33,6 52,8 8,74 -104 6,7 57,5 1,16 4 (-1,5) 7 34,8 53,1 8,76 -105 6,7 57 5,20 5 (-2) 7,2 34,9 53,3 8,76 -105 6,6 56,6 3,81 6 (-3) 7,2 34,1 53,6 7,85 -57 6,6 56 4,76 7 (-3,5) 11,5 43,5 65,9 7,85 -57 3,2 31 4,46 8 (-4) 13,1 41,3 63 7,76 -53 - - 4,20 9 (-5) - 45,2 68 7,74 -52 - - 2,67 10 (-6) - 45,1 67,7 8,65 -99 - - 2,33

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Determination of chlorophyll concentration is needed to estimate primary production of phytoplankton and is the first evidence of a viable food chain. For the analyzed water column were observed fluctuations of the Chl-a concentration, the highest values being recorded between 1.5-4 m depth. Overall, the measured water column falls into the category oligotrophic waters, the euphotic maximum in terms of Chl-a concentration is 1.5 m deep (Marshall and Peters, 1989). Abundance of picoplankton, determined by counting under epifluorescence microscope is shown in Figure 2. Down to 3 m depth no picoplankton was observed, their characteristic autofluorescence signal being detected starting with 3.5 m depth. Their number was relatively low both for algae and cyanobacteria, the most abundant at 4 m depth (6.5 x 10 4 picocyanobacteria /ml and 1.6 x 10 4 picoeukaryotes /ml).

Figure 1. Vertical distribution of physico-chemical parameters in Bottomless Lake (Turda, Cluj County, November 2011).

Figure 2. Abundance picocyanobacteria (PC) and picoeukariotes (Peuk) in water column (3.5 m - 6 m) of Bottomless Lake (Turda, Cluj County)

4 High density of prokaryotic cells was revealed by DAPIU staining (Figure 3). The total cell number ranged between 1.5 x 10 6 cells /ml (-3 m, at the halocline and thermocline) and 2.3 x 10 6 cells /ml (at 1 m depth).

2400000

2200000

2000000

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1600000

Abundenta (cel/ml) 1400000

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1000000 0 -0,5 -1 -1,5 -2 -3 -3,5 -4 -5 -6 Adancime (m) Procariote (archee si bacterii)

Figure 3. Total bacterial cell abundance along water column in Bottomless Lake (Turda, Cluj County) Scientific Conclusions • The data obtained in the experimental part indicated the meromictic nature of Bottomless Lake (Turda), noticing thermal and chemical stratification. • The analyzed the water column falls into the category of oligotrophic waters, the euphotic maximum Chl-a being observed at a depth of 1.5 m. • The estimated number of algae and cyanobacteria was relatively low, the most dense community being observed at 4 m depth (6.5 x 10 4 picocyanobacteria/ ml and 1.6 x 10 4 picoeukariotes/ ml). • Abundance of bacteria is high, without significant fluctuations, the lowest value being recorded at 3 m depth (1.5 x 10 6 cells / ml) and more abundant at 1 meter depth (2.3 x 10 6 cells/ ml).

Managerial Conclusions General scientific objective of this first phase of the project was to locate the sampling sites and conducting preliminary measurements and sampling in the field. The very short time

5 between contract of funding (05.10.2011) and settlement deadline (15.12.2011), added to the problem of receipt of the first installment of funding (14.11.2011) has allowed only one field sampling (03.11.2011 - 06.11.2011, Turda). At this time, however, managerial objective of getting supplies and laboratory reagents was achieved by 100%, ensuring logistics base needed to run the following trips and sampling as well as laboratory analysis for molecular diversity of archaea inhabiting targeted salt lakes. The web page of the project was posted at http://bioge.ubbcluj.ro/grant/Banciu_Horia/

References Clavero, E, Hernandez-Marine, M, Grimalt, JO, Garcia-Pichel, F (2000) Salinity tolerance of diatoms from thalassic hypersaline environments. J. Phycol . 36: 1021-1034. de Leeuw A, Bukowski K, Krijgsman W, Kuiper K (2010) Age of the Badenian salinity crisis; impact of Miocene climate variability on the circum-Mediterranean region. Geology , 38: 715-718. Keresztes K, Somogyi B et al. (2010) Picoplankton in soda lakes of the Carpathian basin. Contrib. Bot . XLV, 41-46. Lewin, RA, Krienitz, L, Goericke, R, Takeda, H, Hepperle, D (2000) Picocystis salinarum gen. et sp. nov.(Chlorophyta) - a new picoplanktonic green alga. Phycologia , 39: 560-565. Marshall, C, Peters, R (1989) General patterns in the seasonal development of chlorophyll a for temperate lakes. Limnol. Oceanogr . 34: 856-867. Ollivier, B, Caumette, P, Garcia, J-L, Mali, RA (1994) Anaerobic bacteria from hypersaline environments. Microbiol. Rev . 58: 27-38. Oren, A (1999) Life at high salt concentrations, In: Dworkin, M, Falkow, S, Rosenberg, E, Schleifer, K-H, Stackebrandt, E. (Eds.), The Prokaryotes, A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications . 3rd. ed. Springer-Verlag, New York. Roesler, CS, Culbertson, CW, Etheridge, SM, Goericke, R, Kiene, RP, Miller, LO, Oremland, RS (2002) Distribution, production, and ecophysiology of Picocystis strain ML from Mono Lake, California.Limnol. Oceanogr. 47: 440-452.

Project Manager, Lect. Dr. Horia Leonard Banciu

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