Eurasia Waste Management Symposium 2011 Bildiri Yazım Kılavuzu

Rogoznica Lake – an Extreme Seawater Environment Hosting Specific Sulfate-reducing Bacterial Community

Milan Čanković*, Ines Petrić and Irena Ciglenečki

Department of Marine and Environmental Research, Ruđer Bošković Institute, Bijenička cesta 54, 10 000 Zagreb *Corresponding author email: [email protected]

Abstract Rogoznica Lake is seasonally stratified and highly eutrophic seawater lake with hypoxic/anoxic layer usually occurring below the depth of 8 m. Due to high phytoplankton activity upper part of the water column is well oxygenated while remineralization processes enhance deposition of organic matter and

nutrients in deeper layer where appearance of hypoxia/anoxia and microbially production of H2S is confirmed. We investigated distribution, diversity and abundance of sulfate-reducing bacteria (SRB) during stratified summer and winter season, by targeting six major phylogenetic groups of SRB using specific 16S rRNA primer sets. Our results implied existence of distinct SRB populations in the water column and sediment. Rarefraction analysis revealed higher diversity of the SRB occupying water layer then the one found in sediments, independent of the sampling season. However, seasonal variations in diversity were observed in the water column and sediment. SRB community was more diverse in winter compared to summer season in water layer while, in opposite, sediment community evolved during summer was more diverse then the one found in winter. Water layer community seems to be more susceptible to changes of physico-chemical parameters, while those in sediment do not follow the same pattern having prorogated response to the changes in the Lake. Low homology of our sequences (as low as 85%) to the sequences in NCBI database further indicated that Rogoznica Lake harbor habitat- specific SRB populations that cannot be associated to known SRB but rather to uncultured bacteria found in extreme marine environments. Keywords: extreme marine environments, Rogoznica Lake, sulfate-reducing bacteria

1. INTRODUCTION Rogoznica Lake, among local inhabitants known as Dragon’s Eye, is a seawater lake situated on the eastern Adriatic coast, 40 km south from Šibenik (Croatia), on the Gradina peninsula (43°32’N 15°58’E). The lake has a circular shape with an area of 10.276m2, maximum length of 143 m, and a maximum depth of 15 m. It is sheltered from the wind by 4–23 m high cliffs that prevent wind-shear mixing (Figure 1). Rogoznica Lake can be characterized as a typical extreme, euxinic environment [1]. The water column is seasonally stratified. Depth of mixolimnion changes seasonally and it is greatly influenced by meteorological conditions, i.e. temperature and rainfall. Vertical mixing usually occurs during winter when cold, oxygen-rich water from the surface sinks downwards [2].

Figure 1. Location, panoramic view (A) and vertical profile (B) of Rogoznica Lake

1 4th International Conference on Research Frontiers in Chalcogen Cycle Science & Technology

From winter to late summer, the less saline top layer is well oxygenated due to very high biological activity

(O2 saturation up to 300%). The more saline bottom layer is enriched with reduced sulfur species, mainly in the form of sulfide which appears there in very high concentration (up to 5 mM) [1-3]. At the oxia-anoxia boundary usually develops the cca 50 cm thick pinky colored chemocline layer due to dense population of purple photoautotrophic sulfide-oxidizing bacteria from genus Chromatium (up to 4.3×108 cells mL-1 in July 1997) that store sulfur intracellularly and comprises up to 51% of total bacteria + [4]. Anoxic deep water is characterized by iodine species (up to 1µM) [5] and nutrients (NH4 , up to 150 µM; 3- 4- -1 PO4 , up to 22 µM; SiO4 , up to 400 µM) [2], [6], as well as dissolved organic carbon (DOC up to 6 mgl ), indicating the pronounced remineralization of allochthonous organic matter in this water layer, produced in the surface water [2], [6], [7]. Sediment is poorly sorted silt and clay silt characterized as authigenic carbonate sediment of mainly biogenic origin with relatively high sedimentation rate (0.093 g/cm3/year). Major mineral is calcite followed by aragonite, quartz, dolomite and pyrite [1]. Although physico-chemical parameters of the lake are well characterized, little is known about the microbial communities of SRB as a main drivers of dissimilatory sulfate reduction process and their fundamental role in biogeochemical sulfur and carbon cycles, given that the eutrophication of the lake is strongly influenced by nutrient recycling under anaerobic conditions. Therefore the aim of this study was to get the deeper insight in distribution, diversity and abundance of SRB in the water layer and sediment of Rogoznica Lake. 1. MATERIALS AND METHODS 1.1. Sampling and Physico-chemical Parameters Measurements Water samples were taken in winter and sammer season of 2014, from anoxic (AN) *12 depth m, sampled in February and August 2014) and chemocline (CC) water layers (between 8-9 m depth, sampled in August 2014). Samples were collected by 5 L Niskin bottle, while the sediments were taken by gravity corer up to the 10 cm depth. Water samples were filtered through 0.22 µm MCE filters and sediment was sliced in surface (SURF) 0-5 cm, and lower (LOW) 5-10 cm sections and frozen till further analysis. Samples for reduced sulfur species (RSS) were analyzed immediately after sampling by electrochemical methods [3],[8]. In each sampling temperature (T), salinity (S), oxygen (O2), pH and redox potential (Eh) were measured in-situ by a HQ40D multimeter probe (Hach Lange, Germany). 1.2. DNA Extraction DNA from filters were extracted (in triplicates) according to a phenol/chloroform procedure [10], and stored at -20°C. Prior to extraction sediment samples were washed with three washing solutions containing Tris- HCl, EDTA and Triton X-100 in order to eliminate extracellular DNA and to enhance the possibility of PCR amplification (REF). Afterwards, DNA from the sediment samples (in triplicates) was extracted according to modified Aurora High Capacity protocol (http://www.borealgenomics.com). Briefly, up to 5 g of sediment was weigh out into 50 ml centrifuge tubes with addition of extraction buffer (Tris-HCl, disodium EDTA, sodium phosphate, all in 100 mM final concentration, 1.5 M NaCl and 1% CTAB) and 250 µl of 20 mg/ml proteinase K. Tubes were incubated at 37°C for 30 min., after which 10% SDS (final concentration) was added and incubated at 65°C for 2 h. After incubation tubes were centrifuged (10 min./6000 g). To the extracted supernatant an equal volume of 24:1 (v/v) chloroform:isoamyl alcohol was added, gently mixed and centrifuged (10 min./1500 x g). Top aqueous layer was taken out and 0.6 x V of isopropanol was added, gently mixed and incubated at room temperature for 1 h. After the centrifugation (30 min./6000 x g) supernatant was discarded and the pellet was washed with 70% ethanol and resuspended in EB buffer. Extracted DNA was additionally purified with PowerClean® DNA Clean-Up Kit (Mo Bio Laboratories,

2 Rogoznica Lake – an extreme environment hosting specific sulfate-reducing bacterial community USA) according to manufacturer’s instructions. Quality and quantity of the extracted DNA were checked spectrofotometrically and on 1% agarose gel (wt/vol) in 10x TBE buffer stained with ethidium bromide. 1.3. PCR PCR primer sets for amplification of the 16S rRNA gene fragments, used in this study were previously designed by Daly et al., 2000 specifically targeting six different phylogenetic groups of SRB [11],. Namely, DFM140/824 (TA-58°C), DBB121/1237 (TA-66°C), DBM169/1006 (TA-64°C), DSB127/1273 (TA-60°C),

DCC305/1165 (TA-65°C) and DSV230/838 (TA-61°C) primer sets were used. PCR amplification was carried out as follows: 95°C for 1 min., annealing at appropriate temperature for 1 min. and 72°C for 1 min. for 40 cycles and additional extension at 72°C for 7 min. Reaction was carried out in total volumeof 25 µl containing: 1.88 µl of each primer (10 µM), 0.2 dNTP mix, 0.1 µl Taq polymerase, 2.5 µl 10x PCR buffer, 13.41 destilled water and 5 µl of DNA template (10 ng/ µl). Although additionally purified, DNA extracted from sediment still contained PCR inhibitors, as indicated by the brown color of the template. In order to circumvent this problem prior to using SRB-specific primers total bacterial 16S rRNA was amplified by using universal 27F/1492R primer set [12]. Afterwards, target groups were amplified by using as a template 2.5 µl of the 16S rRNA product. PCR products were visualized on 1% agarose gel (wt/vol) in 10x TBE buffer stained with ethidium bromide. Bands of the correct size were excised from the gel and purified using GenElute™ Gel Extraction Kit (Sigma-Aldrich, USA) according to manufacturer’s instructions. 1.4. Cloning and Sequencing For phylogenetic studies PCR products purified from the gels were pooled and cloned using the pGEM ®-T Vector System (Promega, France) according to manufacturer’s instructions. In total, 192 positive clones were sent for commercial Sanger sequencing (Macrogen, the Netherlands). Retrieved sequences were edited and checked manually, the closest relatives were identified by BLAST software in the NCBI database. The threshold of 97% similarity was used to define an operational taxonomic unit (OTU). Phylogenetic trees were constructed with ClustalX (v. 1.8). Rooted tree was built using the neighbor-joining method. The robustness of individual branches was estimated by bootstrapping based on 1000 replications. Additionally, to compare sampling completeness and richness between different samples and seasons rarefraction analysis was conducted with the Analytic Rarefaction software (http://www.uga.edu/strata/software/). 1.5. Quantitative PCR (qPCR) qPCR was used to determine the abundance of total bacteria community and phylogenetic groups of SRB. qPCR assays were conducted on ABI 7900 HT Real Time PCR System (Applied Biosystems, USA) in 20 µl final volume containing 10 µl SYBR green PCR Master Mix (Absolute QPCR SYBR Green Rox Abgene, France), 2 µl (10 mM) of each primer and 2 ng of template DNA. For each 16S rRNA target, a standard curve was established using serial dilutions of linearized plasmid (102 to 108 copies) containing gene of interest (bacterial 16S rRNA or 16S rRNA of SRB groups). No-template controls (NTC, n = 2) were also included in all the assays. For the SRB groups specific primers earlier mentioned were used while total bacterial community was assessed by 341f-534r primer set [13]. Samples were run in triplicates. Cycling conditions for DSV subgroup amplification were as follows: 15 min. at 95°C; 35 cycles of 15 s at 95°C, 30 s at 61°C, 30 s at 72 °C. Cycling conditions for DCC subgroup amplification were as follows: 15 min. at 95°C; 35 cycles of 15 s at 95°C, 30 s at 65°C, 30 s at 72 °C. Melting curves were generated after amplification in order to check the specificity of the assays. 2. RESULTS 2.1. Physico-chemical Conditions in Rogoznica Lake The vertical profiles of temperature, salinity and oxygen (Figure 2) clearly show stratified water column with well mixed upper oxygenated A layer above 6 m in both seasons, and more saline andB denser bottom layer. During both seasons CC layer was between 8-9 m, under which anoxic conditions were established.

Figure0 2. Vertical profiles10 of temperature,20 salinity30 and oxygen40 in Rogoznica0 Lake measured10 during20 winter season30 2014. (A)40 and summer0 season 2014 (B) 0

D D e e p p t t h h

( ( m m 10 Temperature (°C) 10 ) ) Salinity Temperature (°C) Oxygen (mg/l) Salinity 12 12 Oxygen (mg/l)

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Bottom layer was enriched in RSS, mainly in the form of sulfide. During summer it reached concentration of 1.3 and 0.35 mM in AN and CC layers, respectively. In winter concentration of sulfide was 0.8 mM in AN layer. 2.2. Presence of Phylogenetically Different SRB Subroups in Samples ‘ Direct’ PCR amplification was attempted with the primers specific for each of the six SRB groups. Desulfotomaculum-like group (DFM), Desulfobulbus-like group (DBB), Desulfobacterium-like group (DBM) and Desulfobacter-like group were not detected in any sample during both seasons. In both, water and sediment samples only two SRB groups were successfully detected corresponding to Desulfococcus- Desulfonema-Desulfosarcina-like and Desulfovibrio-Desulfomicrobium-like group as shown in the Table 1. Table 1. PCR detection of 6 phylogenetically different SRB subgroups in winter and summer season samples by targeting 16SrRNA gene fragment.

Winter 2014. Summer 2014.

Group Water Sedim. Sedim. Water Water Sedim. Sedim. Target Predicted target group gene name (12m) (0-5 cm) (5-10 cm) (8-9 m) (12m) (0-5 cm) (5-10 cm)

16S DFM Desulfotomaculum rRNA ------

16S DBB Desulfobulbus rRNA ------

16S DBM Desulfobacterium rRNA ------

16S DSB Desulfobacter rRNA ------

Desulfonema / 16S DCC Desulfococcus / rRNA + + + - - + + Desulfosarcina

16S Desulfovibrio / DSV rRNA Desulfomicrobium + - - + + - - ‘’+’’ detected; ‘’-‘’ undetected 2.3. Diversity and Phylogenetic Affiliation of SRB populations Figure 3. Diversity and phylogenetic tree constructed by N-J method of DSV subgroup detected in AN (12 m) and CC (8-9 m) layer in winter and summer season. Pies are representing share of total OTUs based on 97% cutoff value.

4 Rogoznica Lake – an extreme environment hosting specific sulfate-reducing bacterial community

CC layer - summer DSV winter AN water layer DSV 80 DSV 92 OTU1 1000 DSV 73 DSV summer CC water layer DSV KK12 OTU2 DSV KK8 1000 Uncultured bacterium clone SPHAL 52 Synechococcus -related DSV summer AN water layer Synecococcus sp KORDI-15 OTU3 DSV 95 DSV 84 OTU4 945 DSV 75 DSV KK22 DSV KK21 OTU5 DSV 78 DSV KK9 OTU6 DSV KK23 DSV KK13 DSV KK7 DSV AN36 DSV AN26 DSV AN34 Summer Cluster 1 DSV AN27 818 DSV KK24 Uncultured δ- AN layer - winter DSV AN46 AN layer - summer DSV AN48 Proteobacterium OTU1 DSV AN34DSV KK10 DSV AN39 OTU1 OTU2 389 DSV AN28 Uncultured bacterium p763 b 4 41 OTU2 OTU3 106 999 Uncultured bacterium PM-150-Bact-09 OTU3 OTU4 Uncultured bacterium C9001C B156 C041 OTU5 885 Uncultured bacterium methane seep Tainan Ridge OTU4 DSV 88 OTU6 OTU5 Uncultured bacterium Ld1 55 Nort Yellow sea sediments OTU7 Uncultured organism SBZO 1841 Guerrero Negro hypersaline 111 OTU6 OTU8 Uncultured_planctomycete_clone_OGT_B4_14 Planctomycete OGT B4 14 hadopelagic sediments Uncultured environmental OTU7 OTU9 Uncultured δ –Proteobacterium MothraB6-68 427 DSV AN42 δ-Proteobacteria 649 DSV KK14 DSV KK4 911 DSVAN40 48 DSV AN45AN37 DSV AN30 0.1 DSV KK17 333 DSV KK11 Uncultured WS3 bacterium clone BFB005 1000 DSV KK15Uncultured division WS3 anoxic fjord Sweden DSV KK6 994 542 DSV KK5 998 DSV AN44 DSV AN41 1000 DSV AN47 DSV AN43 238 998 DSV AN25 DSV 81 769 DSV 94 Winter Sub-cluster Verrucomicrobia bacterium Sylt 22 Verrucomicrobium group Thermodesulfovibrio yellowstonii Verrucomicrobia-related 437 DSV AN31 148 1000 DSV AN48 1 Uncultured environmental DSV AN29 Summer Sub-cluster 1a 1000 Clostridium sp BA-1 Clostridium-related Firmicutes 1000 Clostridiales bacterium r28 DSV 87 DSV 93 529 DSV 85 861 DSV 79 Winter Uncultured Desulfobacteraceae ZLL-D59 Uncultured environmental 639 Uncultured δ-Proteobacteria PET-124 Cluster 1 DSV 91 364 448 DSV 89 Uncultured δ- δ-Proteobacteria 282 DSV 82 566 Proteobacteria Desulfobacterales UnculturedSRB NaphS3δ-Proteobacterium YS-UMF1 C131 Uncultured bacterium JML C01 Uncultured δ -Proteobacterium_SGTA698 1000 DSV 96 DSV 90 Winter 932 Desulfovibrio hydrothermalis AM13 DSV8676 Cluster 2 DSV 77 390 Desulfovibrio sp. An30N-mm Desulfovibrio- Uncultured clone CO1SHNF695 Desulfovibrio salexigens DSM 2638 related 502 1000 Uncultured SRB 2R2V02 DSV AN33 δ-Proteobacteria DSVAN38 DSV AN35 Desulfovibrionales DSVKK20 DSV KK19 Summer DSV KK16 DSV KK3 Cluster 2 DSV KK2 DSV AN32 Desulfomicrobium 993 DSV KK2 1 Desulfomicrobium baculatum HAQ-8 baculatum Desulfomicrobium baculatum 1000 Desulfomicrobium baculatum DSM 4028 954 Desulfomicrobium sp. PR4_C11 995 DSV 74δ-Proteobacterium FM3-F10 Uncultured Desulfuromonadales bacterium clone B154 Winter Cluster 3 δ-Proteobacteria Uncultured bacterium clone MD2894-B23 943 Desulfacter sp. Desulfomonadales- Desulfomonadales Uncultured Synechococcus_A82 1000 Synechococcus sp. RS9918 related 845 Synechococcus sp._RS9918 Uncultured bacterium J608-77 Synechococcus sp. RCC36 Uncultured bacterium 014-12FFSCA001-A1B1 stratified seawater lake Pseudomonas aeruginosa strain DSM 50071T Sequence analysis of the DSV clone libraries obtained from the water layer sample suggested high diversity of the DSV subgroups (Figure 3.). Retrieved sequences belonged to three major polygenetic groups (i.e. δ- Proteobacteria, Verrucomicrobium group and Firmicutes). Season specific clustering was likewise observed. Sequences from AN layer in winter season grouped in 10 OTUs (each with up to 3 representatives), majority of which was uncultured δ-Proteobacteria (90-91% homology), followed by Desulfovibrionales-related group (91- 96% homology) and Desufomonadales- and Verrucomicrobium-related group (91% homology). 4th International Conference on Research Frontiers in Chalcogen Cycle Science & Technology Lower diversity of the clone libraries was found during summer season represented by 6 and 7 OTUs in CC and AN layer, respectively. Majority of sequences in CC layer was related to uncultured environmental δ- Proteobacteria (94-97% homology), followed by Desulfovibrionales-related group (91-96% homology). In AN layer dominant OTU was similar to CC layer with dominant sequences belonging to uncultured environmental δ-Proteobacteria (94-95% homology), followed by Desulfovibrionales-related group (85% homology), with exception of OTU related to Firmicutes (Clostridium)-related group (96% homology). Synecoccocus (Cyanobacteria)-related group was also detected by this primer pair (90-99% homology). DCC SRB subgroup was detected only in AN water layer in winter seasons. This subgroup evolved during winter season showed sequences grouping in 10 OTUs. All of retrieved sequences were related to uncultured δ-Proteobacteria, Desulfosarcina- related (Desulfobacterales), showing homology of 92-98%. Sequence analysis showed that sediment samples clearly differentiated from water samples. In sediment samples only DCC subgroups were detected. Retrieved clone sequences, diversity and phylogenetic affiliation of SURF and LOW winter sediment samples are shown in Figure 4. Figure 4. Diversity and phylogenetic tree constructed by N-J method of DCC subgroup detected in SURF (0-5 cm) and LOW (5-10 cm) winter sediment layer. Pies are representing share of total OTUs based on 97% cutoff value.

6 Rogoznica Lake – an extreme environment hosting specific sulfate-reducing bacterial community Phylogenetic affiliations of DCC 16SrRNA clone libraries of the sediment microbial community found in the winter season is relatively clearly layering between SURF and LOW samples. Diversity is quite lower than the one found in the water samples, with sequences gouping in only 3 OTUs at each depth. Half of the SURF (0-5 cm) samples sequnces (OTU3) were Desulfosarcina (Desulfobacterales)-related group (97-99% homology) while the rest (OUT1 and 2) corresponded to uncultured environmental δ-Proteobacteria (97-98% homology to uncultured sea-sediment bacteria). SRB community found in LOW (5-10 cm) samples comprised more than 83% (OTU1 and 2) sequences related to uncultured environmental δ-Proteobacteria (98-99% homology to uncultured sea-sediment bacteria), while the rest of the sequences were Desulfosarcina (Desulfobacterales)-related group (99% homology) (OTU3). Analysis of summer sediment samples also showed that only DCC subgroups were present. However, in this season no clear layering between SURF and LOW samples was seen. Based on the observed clustering it is visible that the diversity of SRB communities was generally higher in summer season compared to winter samples, with majority of sequences being correlated to the uncultured environmental δ-Proteobacteria. SURF (0-5 cm) sequences grouped in 4 OTUs, while those from LOW (5- 10 cm) samples grouped in 7 OTUs. All clones from SURF samples were related to uncultured environmental δ-Proteobacteria (98-99% homology to uncultured sea-sediment bacteria). Majority of LOW sample clones were related to uncultured environmental Desulfobacteriaceae (98-99% homology), followed by uncultured environmental δ-Proteobacteria (97-98% homology to uncultured sea-sediment bacteria) and uncharacterized marine bacterium and Desulfosarcina (Desulfobacterales)-related group (98% homology). 2.4. Abundance of the Detected SRB Subgroups Quantification of 16S rRNA target gene by using primer sets specific DCC and DSV SRB subgroups allowed us to determine abundances of these populations in total microbial community. 2.4.1. Abundance of SRB in Water Column Gene copy number of the total bacterial 16S RNA and 16S rRNA of the DVS and DCC SRB subgroups is shown in Figure 5. As visible total bacteria were more abundant in water layer during the winter season (average 9.53 x 106 gene copy number/ng DNA), while in summer their abundances reached average of 6.56 x 105 and 7.38 x 105 gene copy number/ng DNA in CC and AN layer, respectively. Figure 5.Copy number of total bacterial 16S rRNA and 16S rRNA of detected SRB subgroups (DSV and DCC) in water layer sampled during winter and summer seasons

CC = chemocline layer (8-9 m); AN = anoxic layer (12 m). DSV and DCC SRB populations were shown to be represented in small percentage in the total bacterial community. In winter season they accounted only 0.2 and 2.3% (DSV and DCC, respectively) of the total community in AN layer. During the summer season DSV subgroups, comprised 0.5% and 1.9% in AN layer and 0.3% and 1.7% (for DSV and DCC, respectively) in CC layer. 2.4.2. Abundance of SRB in Sediment Abundances of the 16S rRNA targeted gene in total bacteria and in DCC SRB population are shown in Figure 6. As visible total bacterial abundance varied only slightly between the seasons (5.03 x 10 5 to 3.39 x 105 gene copy number/ng DNA in winter SURF and summer LOW, respectively). DCC subgroup was comprised from 1.9% up to 5.2% of total 16S rRNA in summer LOW and summer SURF layer, respectively. Figure 6. Copy number of total bacterial 16S rRNA and 16S rRNA of DCC SRB subgroup in sediment sampled during winter and summer season 4th International Conference on Research Frontiers in Chalcogen Cycle Science & Technology

SURF = surface 0-5 cm sediment; LOW = lower 5-10 cm sediment 2.4.3. Rarefraction Analysis Rarefraction analysis was conducted for all samples with results presented in Figure 7. Figure 7. Rarefraction analysis conducted with the Analytic Rarefaction software (http://www.uga.edu/strata/software/).

12 Δ DSV anoxic water layer winter Χ DCC anoxic water layer winter Ɣ DSV anoxic water layer summer 10 Ƒ DSV chemocline water layer summer

m 4 u N

2 Ɣ DCC 0-5 cm sediment winter ж DCC 5-10 cm sediment winter ▄ DCC 0-5 cm sediment summer Ÿ DCC 5-10 cm sediment summerr 0 0 5 10 15 20 25

Number of OTUs group members

From the rarefaction curves obtained, only those from winter AN layer did not reached an assymptote, indicating that the diversity of theese SRB communities was not fully recovered by the sampling effort. Curves showed that the relative richness was different in different sampling sesasons as well as from the samples collected from various water (AN and CC) and sediment (SURF and LOW) layers. 3. DISCUSSION By using 16S rRNA as a molecular marker we aimed to characterize SRB bacterial community in Rogoznica Lake being a main driver of the sulfate reduction process in this seawater ecosystem. PCR-results suggested presence of two subgroups of SRB, DSV and DCC, implying that bacteria closely related to Desulfonema/Desulfococcus/Desulfosarcina-group and Desulfovibrio/Desulfomicrobium-group represent SRB community of the lake . However, phylogenetic analyses revealed that SRB community existing in anoxic water layer as well as in anoxic sediment is rather unique and that majority of the sequences did not match well (85-98% homology) with known SRB bacteria but rather showed homology to the uncultured bacteria of the δ-Proteobacteria phyla, found in different marine and polluted environments. This was confirmed by the qPCR analysis showing that by using these target genes we were able to characterize only a small fraction of the total SRB community therefore further analysis are required based on some other, most likely functional dsrAB gene, which would give more accurate estimation of the abundance of this community in Rogoznica Lake. 16S rRNA clone libraries revealed clear differences in the structure of the SRB communities across vertical profile of the Lake, from CC water layer to deeper sediment with a lmost complete shift in the composition of SRB assemblages between water and sediment layers. Several bacterial populations seemed to apear in the targeted community in correlation with the sampling season. Two SRB

8 Rogoznica Lake – an extreme environment hosting specific sulfate-reducing bacterial community populations, clustering with Desulfomicrobium baculatum (Desulfovibrionales) (in CC and AN samples) and Clostridium (Firmicutes) (in AN layer) were found exclusively in water layer during summer sampling. During colder winter season members of Desulfomonadales, Desulfovibrionales, Desulfobacterales and Verrucomicrobium groups appeared in the AN water layer. Likewise, SRB population related to Desulfosarcina genera were detected only during winter in the AN layer. These organisms are known to oxidize a diverse range of organic compounds including acetate and other short-chain fatty acids to CO 2. Sequences from this group have also been recovered from other marine and freshwater environments [16-18]. Desulfovibrionales and Verrucomucrobium bacteria are also known to oxidize simple molecules such as lactate, pyruvate, malate to CO2. Rarefraction analysis further confirmed differeneces between water layers and sediment. We observed that SRB community occupying water layer maintained higher diversity throughout the year when compared to the one found in sediments. In addition, in summer water layer, in the conditions of higher hydrogen sulfide concentrations, temperature and total organic carbon values (data not shown), SRB community was less diverse then in the winter water layer probably due to community enrichment by highly adapted sub-population of SRB. Population dynamics observed for the sediments, where diversity was higher during summer season, could not be explained in the same manner indicating that the processes in the sediment, known to be slower and less susceptible to variation of physico-chemical and biological conditions, do not follow the same pattern having prorogated response to the same changes in the water column of the Lake. It has been seen previously that extremely variable S, T, concentration of nutrients, mixing, redox and euxinic conditions can highly influence phytoplankton and zooplankton community structure in the upper layers of Rogoznica lake [14]. Likewise, study on bacterial community occupying this oxigenated layer suggested that its structure can be strongly affected by changes in phyciso-chemical parameters in the lake [15]. This community shifted from anoxygenic phototrophic sulfur bacteria populations to gammaproteobacterial sulfur oxidizers as a result of water layer mixing followed by anoxic holomictic event. Although it is known that lower layer of the lake is less susceptible to changes in physico- chemical parameters, confirmed by the measurements during the investigated seasons (T, S and pH varried only slighty in the anoxic layer of the lake), we cannot completely exclude them as being responsible for the observed shifts in the SRB community structure. It is more likely, that dynamics of the phytoplankton community during the year would affect community in deeper layers of the lake. Different composition of organic matter, supplied from this oxygenated upper layer, could be coupled to the changes in the SRB community structure. Simple organic molecules are presumably oxydized above and in the CC and AN layers, while the more complex and refractory fraction probably exists in the prebottom and sediment [1],9], where it could be oxydized by different SRB. 4. CONCLUSION This study explored for the first time specific anoxic SRB community in Rogoznica lake. Results gave new insight on the diversity, abundance and phylogeny of SRB. Althouh such extreme enviroments were once considered as ‘’dead zones’’, from the microbiologically point of view, our results suggested that this ecosystem represent quite vivid area. Results clearly showed distinct seasonal variations in diversity and abundance of SRB communities residing in the water column as well as in the sediment. Low sequence homology to cultured SRB indicated presence of specific SRB community in the lake even on high taxonomical level of phylum. Although small and isolated, seawater system of Rogoznica Lake represents higly productive ecosystem. Therefore, data presented here show a complex SRB distribution and diversity supporting the idea that habitat-specific SRB communities contribute to the anaerobic degradation of organic matter in the Lake as well as the cycling of sulfur and carbon species. REFERENCES [1] Ciglenečki I, Pichler S, Prohić E, Ćosović B. Distribution of redox-sensitive elements in bottom waters, porewaters and sediments of Rogoznica Lake (Croatia) in both oxic and anoxic conditions. Water Air Soil Poll 2006; 6:537-545. [2] Ciglenečki I, Carić M, Kršinić F, Viličić D, Ćosović B. 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