Spatio-temporal dynamics of sulfate-reducing bacteria in extreme environment of Rogoznica Lake revealed by 16S rRNA analysis

Journal of Marine Systems 172 (2017) 14–23

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Spatio-temporal dynamics of sulfate-reducing bacteria in extreme environment of Rogoznica Lake revealed by 16S rRNA analysis Milan Čanković, Ines Petrić ⁎, Marija Marguš, Irena Ciglenečki Division for Marine and Environmental Research, Ruđer Bošković Institute, Bijenic street 54, 10 000 Zagreb, Croatia

a r t i c l e

i n f o

Article history: Received 7 December 2016 Received in revised form 1 March 2017 Accepted 3 March 2017 Available online 6 March 2017 Keywords: Sulfate-reducing bacteria 16S rRNA gene Community shifts Extreme marine environments Euxinic Rogoznica Lake

a b s t r a c t Highly eutrophic and euxinic seawater system of Rogoznica Lake (Croatia) was used as a study site for investigation of distribution, diversity and abundance of sulfate-reducing bacteria (SRB) during stratified conditions in the summer and winter season, by targeting 6 phylogenetic subgroups of SRB. 16S rRNA gene sequences indicated that community cannot be directly related to cultured SRB species but rather that Rogoznica Lake harbors habitat-specific SRB populations associated to bacteria belonging to δ-Proteobacteria with few Firmicutes and Verrucomicrobium-related populations. Clear spatial-temporal shifts in the SRB community structure were observed. Results implied existence of distinct SRB populations between the water column and sediment, as well as higher diversity of the SRB occupying water layer then the ones found in the sediment. Likewise, seasonal variations in populations were observed. While SRB community was more diverse in the winter compared to the summer season in the water layer, situation was opposite in the sediment. Water layer communities seem to be more susceptible to changes of physico-chemical parameters, while those in the sediment have prorogated response to these changes. Results indicate that SRB diversity is still highly underestimated in natural environments, especially in specific habitats such as Rogoznica Lake. Presented data show a complex SRB diversity and distribution supporting the idea that habitat-specific SRB communities are important part of the anaerobic food chain in degradation of organic matter as well as cycling of sulfur and carbon species in the Lake and similar anoxic environment. © 2017 Published by Elsevier B.V.

1. Introduction Rogoznica Lake, known as Dragon's Eye, is a highly eutrophic seawater lake situated on the eastern Adriatic coast (Ciglenečki et al., 2015). With no fresh water inflow or drifts Lake can be considered as a self-sustainable environment. This unique ecosystem represents an extreme environment with euxinic (i.e. sulfidic) and meromictic characteristics (Bura-Nakić et al., 2009; Ciglenečki et al., 2006, 2005). In meromictic ecosystems stratification of the water column is maintained throughout the year. Reduced vertical mixing usually results in anoxia in deeper water layers and accumulation of sulfide, nutrients and organic matter (OM) in the monimolimnion (Romero and Melack, 1996). Similarly, in Rogoznica Lake oxic surface water layer (mixolimnion) and anoxic deeper water layer (monimolimnion) prevail during the year. At the interface between the mixolimnion and the monimolimnion, a steep chemical gradient called a chemocline is formed (Kubo et al., 2014). In rare occasions, depending on the meteorological conditions, anoxic holomictic conditions in the whole water column can occur, causing ⁎ Corresponding author. E-mail addresses: [email protected] (M. Čanković), [email protected] (I. Petrić), [email protected] (M. Marguš), [email protected] (I. Ciglenečki).

http://dx.doi.org/10.1016/j.jmarsys.2017.03.003 0924-7963/© 2017 Published by Elsevier B.V.

mass mortality of all pelagic and benthic eukaryotes (Ciglenečki et al., 2005, 2015; Kršinić et al., 2000). Out of all lakes less than 1% (Black Sea, Baltic Sea, Norwegian fjords, anoxic brines of Eastern Mediterrannean, Orca basin, Cariaco Basin, lakes Pavin and Annecy etc.) are described as meromictic (Baatar et al., 2016; Gugliandolo et al., 2011; Izdar, 1991; Millero, 1991; Scranton et al., 2006; Van Cappellen et al., 1998; Vanderweijden et al., 1990; Lehours et al., 2005). Due to the high vertical stability of the water masses and physicochemical gradients (in particular oxygen) meromictic lakes with its anoxic layer provide a suitable environment for development of sulfatereducing bacteria (SRB) (Inceoglu et al., 2015; Kubo et al., 2014). SRB are commonly encountered in freshwater sediments, anoxic waters, soils, biofilms, intestinal contents and especially in marine sediments where they can account for N 50% of the organic carbon mineralization (Jørgensen, 1978; Postgate, 1984; Widdel, 1988). SRB, representing large and extremely diverse physiological group, use sulfate as an oxidant for the degradation of organic matter (Lehours et al., 2005; Marschall et al., 1993; Teske et al., 1996). They have the ability to grow on various substrates, such as lactate, carbon monoxide, methanol, amino acids and sugars (Postgate, 1984; Baena et al., 1998; Henstra et al., 2007; Nazina et al., 1988; Ollivier et al., 1988; Parshina et al., 2005; Sass et al., 2002; Stams et al., 1985). Currently, sulfate reducers can be

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divided into two main groups: those that degrade organic compounds incompletely to acetate and those that degrade organic compounds completely to carbon dioxide (Muyzer and Stams, 2008). Up to date 60 genera containing 220 species of SRB are known (Barton and Fauque, 2009). Due to its specificity i.e. both euxinic and meromictic features, Rogoznica Lake can be considered as a natural bioreactor i.e. laboratory to study microbial processes in the redox transition zones, including those generated by SRB. Although physico-chemical and some microbiological parameters of the Lake have been studied over the past years (Ciglenečki et al., 2005; Pjevac et al., 2014), nothing is still known about the microbial communities of SRB harboring hypoxic/anoxic niche of the Lake. Diversity of microbial communities, including SRB, have been focus of many studies conducted in different hypoxic and anoxic aquatic environments (Baatar et al., 2016; Inceoglu et al., 2015; Jørgensen, 1978). However, studies on the SRB spatial-temporal distribution and comparison between communities in the sediment and the overlying water column are still rare (Bosshard et al., 2000; Koizumi et al., 2004; Kondo and Butani, 2007). Therefore, the aim of this study was to get better insight into biogeochemical processes in the unique ecosystem of Rogoznica Lake by following behaviour of SRB in the whole hypoxic/anoxic niche during summer and winter season. These organisms are the main drivers of dissimilatory sulfate reduction process occurring both in the water layers and sediment of the Lake. Using 16S rRNA as a gene marker we targeted 6 phylogenetic subgroups of SRB and studied their distribution, diversity and abundance. By using culture-independent methodology i.e. amplifying marker gene directly from the environment we aimed to unfold true diversity of SRB in Rogoznica Lake. The results of this study will add to our understanding of the role and behaviour of SRB communities in similar eutrophic and euxinic ecosystems. 2. Materials and methods 2.1. Study site Rogoznica Lake is situated on the eastern Adriatic coast (Croatia), on the Gradina peninsula (43°32′N 15°58′E) (Fig. 1). The Lake has a circular shape with an area of 10.276 m2, maximum length of 143 m, and a maximum depth of 15 m. It is sheltered by 4–23 m high cliffs that prevent wind-shear mixing. Chemocline is characterized by the presence of the sulfide-oxidizing phototrophic bacteria from genus Chromatium (up to 108 cells ml−1)

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(Ciglenečki et al., 1998; Pjevac et al., 2014). Depth of chemocline is changing daily and seasonally, greatly influenced by meteorological conditions. Monimolimnion is characterized by relatively high concentrations of 3− iodine species (up to 1 μmol), nutrients (NH+ 4 , up to 150 μmol; PO4 , up , up to 400 μmol), dissolved organic carbon (DOC up to to 22 μmol; SiO4− 4 6 mg l−1) and reduced sulfur species (RSS), mainly in the form of sulfide which appears in very high concentration (up to 5 mmol) (Ciglenečki et al., 2015; Ćosović et al., 2000; Žic et al., 2013). These parameters indicate the pronounced remineralization of allochthonous organic matter produced in the mixolimnion, and activity of SRB as main sulfide producers. Sediment in Rogoznica Lake is poorly sorted silt and clay silt characterized as authigenic carbonate sediment of mainly biogenic origin. Major mineral is calcite followed by aragonite, quartz, dolomite and pyrite (Ciglenečki et al., 2006). Sediment is laminated indicating no bioturbation or any other physical forces that can cause mixing. 2.2. Sampling and measurements of physico-chemical parameters Water samples were taken in winter and summer season from the anoxic (12 m depth, sampled in February and August 2014) and the chemocline water layers (between 8 and 9 m depth, sampled in August 2014) from the middle of the Lake. Water samples were collected by 5 l Niskin bottle and filtered (0.22 μm MCE) within a few hours after sampling. Sediments were taken by gravity corer (up to the 10 cm) and sliced in surface (SURF) 0–5 cm, and lower (LOW) 5–10 cm sections and frozen (−20 °C) till further analysis. All samples were taken in triplicates. Samples for reduced sulfur species (RSS) were analyzed immediately after sampling by electrochemical method (Bura-Nakić et al., 2009; Ciglenečki and Ćosović, 1997). Samples for dissolved and particulated organic carbon (DOC and POC), previously filtered, were determined by standard high temperature catalytic oxidation method (TOC-VCPH-5000 solid sample TOC analyzer), (Shimadzu, Japan). Temperature (T), salinity (S), dissolved oxygen (O2) and pH were measured in-situ by a HQ40D multimeter probe (Hach Lange, Germany). 2.3. DNA extraction DNA from filters was extracted according to a phenol/chloroform procedure (Massana et al., 1997). Prior to DNA extraction from the 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 (Fortin et al.,

Fig. 1. Location, panoramic view (a) and vertical profile (b) of Rogoznica Lake.

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2004). DNA from sediment was extracted according to modified Aurora High Capacity protocol (http://www.borealgenomics.com). Briefly, up to 5 g of sediment was mixed with 13.5 ml of extraction buffer (TrisHCl, disodium EDTA, sodium phosphate, all in 100 mmol final concentration, 1.5 mol NaCl and 1% CTAB) and 250 μl of 20 mg/ml proteinase K and incubated at 37 °C for 30 min, after which 10% SDS was added, incubated at 65 °C for 2 h and centrifuged (10 min/6000 g). An equal volume of 24:1 (v/v) chloroform:isoamyl alcohol was added to the supernatant, gently mixed and centrifuged (10 min/1500 × g). Top aqueous layer was taken out and 0.6 xV of isopropanol was added, gently mixed and incubated at room temperature for 1 h. After the centrifugation (30 min/6000 ×g) supernatant was discarded and the pellet was washed with 70% ethanol and resuspended in elution buffer. Extracted DNA was additionally purified with PowerClean® DNA Clean-Up Kit (Mo Bio Laboratories, USA) according to manufacturer's instructions. Quantity and quality of the extracted DNA were checked spectrophotometrically (by measuring the absorbance at 260 nm) and on 1% agarose gel (wt/vol). 2.4. PCR amplification of 16S rRNA genes 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 subgroups of SRB (Desulfotomaculum-like group, DFM140/824, TA-58 °C; Desulfobulbuslike group, DBB121/1237, TA-66 °C; Desulfobacterium-like group, DBM169/1006, TA-64 °C; Desulfobacter-like group, DSB127/1273, TA60 °C; Desulfonema/Desulfococcus/Desulfosarcina, DCC305/1165, TA65 °C and Desulfovibrio/Desulfomicrobium DSV230/838; TA-61 °C) (Table 1). PCR amplification was carried out at 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. Although additionally purified, DNA extracted from sediment still contained PCR inhibitors, as indicated by the brownish color of the template. Therefore, prior to using SRB-specific primers total bacterial 16S rRNA was amplified by universal 27F/1492R primer set as previously described (Lane, 1991). Afterwards, target groups were amplified by using 2.5 μl of the 16S rRNA product as a template. 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.

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. 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/). The nucleotide sequence data reported are available in the NCBI database under accession numbers KT424312-KT424494. 2.6. qPCR analysis of total bacterial community and targeted populations To verify the absence of inhibitors in our DNA extracts, inhibition test was conducted. A known amount of the plasmid pGEM-T Easy Vector (Promega, France) was mixed with the DNA extracts or water before running a qPCR with plasmid-specific T7 and SP6 primers as previously described (Henry et al., 2004). The measured cycle threshold (Ct) values obtained for the different DNA extracts and for the water controls were not significantly different, indicating that no inhibition occurred. qPCR was used to determine the abundance of total bacterial communities and phylogenetic subgroups 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 or SRB subgroups 16S rRNA). No-template controls (NTC, n = 2) were also included in all assays. For SRB subgroups specific primers earlier mentioned were used, while the total bacterial community was assessed by 341f534r primer set (Henry et al., 2004). Samples were run in triplicates. Cycling conditions for DSV subgroup amplification were: 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: 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 assays. 3. Results

2.5. Cloning and sequencing of the target gene

3.1. Physico-chemical conditions in the water column of Rogoznica Lake

For phylogenetic studies PCR products purified from the gel were 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%

The vertical profiles of temperature, salinity, pH, oxygen and RSS (Fig. 2) clearly showed stratified water column with mixolimnion above 8 m depth in both seasons, and more saline, denser and anoxic monimolimnion beneath. During both seasons chemocline layer was situated between 8 and 9 m under which anoxic conditions were established.

Table 1 PCR detection of 6 phylogenetically different SRB subgroups in the winter and summer season by targeting 16S rRNA gene fragment. Winter 2014

Summer 2014

Target gene

Group name

Predicted target group

Water (12 m)

Sediment (0–5 cm)

Sediment (5–10 cm)

Water (12 m)

Water (8–9 m)

Sediment (0–5 cm)

Sediment (5–10 cm)

16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA 16S rRNA

DFM

Desulfotomaculum

−

−

−

−

−

−

−

DBB

Desulfobulbus

−

−

−

−

−

−

−

DBM

Desulfobacterium

−

−

−

−

−

−

−

DSB

Desulfobacter

−

−

−

−

−

−

−

DCC

Desulfonema/Desulfococcus/Desulfosarcina +

+

+

−

−

+

+

DSV

Desulfovibrio/Desulfomicrobium

−

−

+

+

−

−

+

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Fig. 2. Vertical profiles of temperature [°C], salinity, pH, oxygen [mg/l] and RSS [mol/l] (lower axis) in Rogoznica Lake measured during the winter (a) and summer season (b) of 2014.

Temperature of the monimolimnion at sampling depths ranged from 15 °C to 18.3 °C in the winter and summer season, respectively. Salinity ranged from 29.7 during the winter to 34.7 in the summer season. pH was relatively constant throughout the year in the monimolimnion (7.05 to 7.17, in the summer and winter season, respectively), which indicates relative stagnation of temperature and pH in anoxic layer. Monimolimnion, i.e. bottom layer was enriched in RSS (here defined as compounds that contain sulfur in the oxidation states − 2 and 0), mainly in the form of sulfide. During the summer it reached concentration of 1.3 and 0.35 mmol in anoxic and chemocline layers, respectively. In the winter, concentration of sulfide dropped to 0.8 mmol in the anoxic layer. During summer sampling DOC was measured in the concentrations of 2.7 (chemocline) and 3.11 mg/l (anoxic water layer) while POC was 3.7 (chemocline) and 2.54 mg/l (anoxic water layer). During colder season these values corresponded to 1.1 mg/l (chemocline) and 2.48 mg/l (anoxic water layer) for DOC and 1.26 mg/l (chemocline) and 1.98 mg/l for POC (anoxic water layer) measurements. Vertical profiles of DOC and POC are given in the supplementary materials (Fig. S1).

S1b were likewise affiliated to δ-Proteobacteria but were limited only to the anoxic layer and were less abundant. Population S5 clustering with Clostridium (Firmicutes) was also limited to this anoxic layer. Retrieved clone marked as S2 was detected only in the chemocline layer. Population S3, found in both the anoxic and chemocline layers, was shown to cluster with Desulfomicrobium baculatum. The most unique population, due to the low homology of only 85%, was the one marked as S4, representing sequences related to Desulfomicrobium bacterium. Cluster designated as uncultured δ-Proteobacteria population (W2) found in the winter samples contained sequences phylogentically different from those uncultured δ-Protobacteria found in the summer. This population was found to be the most abundant in the winter samples. Population W1 clustered with Desulfobacterales/Desulfosarcina group. Desulfobacterium (W3) and Desulfovibrio (W4) were detected only in the winter season. The lowest sequences abundance was associated to Verrucomicrobia-related group (W5) found exclusively in the winter season. 3.4. Community structure, dynamics and phylogenetic affiliations in sediment samples

3.2. Presence of phylogenetically different SRB subgroups in samples PCR amplification was attempted with the primers specific for each of 6 SRB subgroups. Desulfotomaculum-like group (DFM), Desulfobulbus-like group (DBB), Desulfobacterium-like group (DBM) and Desulfobacter-like subgroups (DSB) were not detected in any sample. In both, water and sediment samples only two SRB subgroups were successfully detected corresponding to Desulfococcus/Desulfonema/Desulfosarcina-like and Desulfovibrio/Desulfomicrobium-like subgroup as shown in Table 1. In the winter season DCC subgroup was detected in anoxic water layer as well as at both sampling depths in sediment samples, while DSV subgroup was detected only in anoxic water layer. During the summer season DCC subgroup was present only in the sediment, while DSV subgroup was detected only in sampled water layers. 3.3. Community structure, dynamics and phylogenetic affiliations in the water samples

Phylogenetic trees of retrieved sequences from the sediment, from the summer and winter seasons are presented in Fig. 3b. Population C1 was the most abundant in the summer season compared to the winter season, showing phylogenetic affiliation with uncultured δProteobacteria. In contrast, populations C2 were more abundant in the winter and were found throughout the sediment, clustering with Desulfosarcina-related group. Populations marked as C3, affiliating with uncultured δ-Proteobacteria were lowly represented in samples. They were found during both seasons, but only in the lower sediment. In contrast, populations S2 and W1 were found only in the surface sediment showing higher abundance in the summer season. Populations S3 and S4 showed to be unique for the summer lower sediment clustering with uncultured marine bacterium. The most abundant (W2) population was found exclusively in the winter lower sediment samples, affiliating with uncultured δ-Proteobacteria. 3.5. Diversity of SRB in Rogoznica Lake

Comparison between SRB community phylogenies found in summer and winter seasons are presented in Fig. 3a. Retrieved sequences marked as S1a correspond to the most abundant population found exclusively in the summer anoxic and chemocline water samples showing phylogenetic affiliations with uncultured δ-Proteobacteria. Populations

Based on the 97% threshold value all retrieved sequences were additionally grouped into operational taxonomy units (OTUs). Sequences retrieved from the water layer could be associated to 3 major phylogenetic groups i.e. δ-Proteobacteria, Verrucomicrobia group and Firmicutes.

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M. Čanković et al. / Journal of Marine Systems 172 (2017) 14–23

Sequences from the anoxic layer in the winter season were grouped in 12 OTUs, majority of which represented uncultured δ-Proteobacteria (90– 91% homology), followed by Desulfovibrionales-related group (91–96% homology), Desulfosarcina-, Desulfobacterium- and Verrucomicrobia-related group (91% homology). Lower diversity of clone libraries was found during the summer season (6 OTUs in the chemocline and 7 OTUs in the anoxic layer). Sequences retrieved from the sediment samples were found to be phylogenetically different from those in the water, showing better homology to correlated NCBI sequences. Sequences were grouped in 3 OTUs at both sampled depth in the summer season and 7 OTUs in the lower sediment in the winter, with majority of sequences correlating to uncultured environmental δ-Proteobacteria as shown in Table 2. Rarefaction curves of 5 out of 8 samples did not reached an asymptote at 3% distance cutoff level indicating that the diversity of these SRB communities was not fully recovered by the sampling effort, highlighting the need for further investigation (Supplementary material, Fig. S2). This was especially evident for the water layer samples.

Diversity was shown to be different in different sampling seasons as well as between samples collected from the water (anoxic and chemocline) and sediment (surface and lower) layers.

3.6. Abundance of SRB in water samples Quantification of 16S rRNA target gene by using primer sets specific for DCC and DSV SRB subgroups allowed us to determine their abundances in total microbial community. Gene copy numbers of total bacterial 16S rRNA and 16S rRNA of DVS and DCC SRB subgroups are shown in Fig. 4a. Total bacteria in the water layer during the winter season accounted an average of 9.53 × 106 ± 2.01 × 107 gene copy number/ ng DNA, while in summer their abundances reached average of 6.56 × 105 ± 4.96 × 105 and 7.38 × 105 ± 8.07 × 105 gene copy number/ng DNA in chemocline and anoxic layers, respectively. Kruskal-Wallis test (P b 0.05) showed no significant differences among the total bacterial community abundances.

Fig. 3. Summer (left) and winter (right) season phylogenetic tree showing phylogenetic affiliations of 16S rRNA gene sequences retrieved from water samples (a) and sediment samples (b). Trees were constructed by N-J method and the robustness of individual branches was estimated by bootstrapping based on 1000 replications. Environmental sequences determined in this study are shown in bold. Horizontal scale bars represent genetic distance. Different rectangles are representing different populations. S-clusters corresponding to the summer season; W-clusters corresponding to the winter season; C-clusters corresponding to both seasons.

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Fig. 3 (continued).

DSV and DCC SRB populations contributed a small portion of the total bacterial community. In the winter season they accounted only 0.2% and 2.3% (DSV and DCC, respectively) of the total community in the anoxic layer. During the summer only DSV subgroup was detected, comprising 0.5% in the anoxic and 0.3% in the chemocline layer. Kruskal-Wallis test (P b 0.05) showed significant differences among the DSV winter anoxic and summer chemocline populations (Fig. 4a).

3.7. Abundance of SRB in the sediment Abundances of 16S rRNA gene in the total bacteria and of DCC SRB population are shown in Fig. 4b. Total bacterial abundance did not vary significantly (Kruskal-Wallis test, P b 0.05) between seasons (5.03 × 105 ± 9.54 × 104 to 3.39 × 105 ± 1.08 × 105 gene copy number/ng DNA in winter surface and summer lower sediment, respectively). DCC subgroup comprised from 1.9% up to 5.2% of total 16S rRNA in the summer lower and summer surface sediment layer, respectively. Kruskal-Wallis test (P b 0.05) showed no significant differences among the winter and summer DCC samples while differences were observed in the summer between upper and lower sediment samples (Fig. 4b).

4. Discussion So far, little is known about the abundance and diversity of SRB that thrive in the unusual ecosystem of Rogoznica Lake. Phylogenetic analysis based on 16S rRNA gene marker revealed that SRB are present across the whole studied vertical profile of the Lake indicating that dissimilatory sulfate reduction process occurs from the chemocline water layer down to the sediment. These results further confirmed that measured RSS species are result of biological processes governed by SRB. PCR analysis targeting phylogenetic subgroups of SRB suggested that Desulfonema/Desulfococcus/Desulfosarcina-group and Desulfovibrio/ Desulfomicrobium-group represent dominant SRB in the Lake. However, sequences retrieved from the water layer indicated that our community cannot be directly related to known SRB strains but rather that the Lake harbors specific SRB populations associated on a higher taxonomic level to δ-Proteobacteria with few Firmicutes and Verrucomicrobium-related populations found in different marine and polluted environments. This was even more profound in the sediment. Our study suggested that the diversity of SRB is still highly underestimated in natural environments. Detailed analysis of clone libraries further revealed differences in the structure of SRB communities across the vertical profile of the Lake with clear shift in the composition of SRB assemblages between water and

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Table 2 Operational taxonomy units (OTUs) and phylogenetic affiliation of SRB based on 16s rRNA gene retrieved from Rogoznica Lake. Sample

Phylogenetic affiliationa

No. of OTUsb

No. of clones

Homology (%)c

Summer CCd layer

Uncultured environmental δ-Proteobacteria δ-Proteobacteria/Desulfovibrionales Desulfomicrobium baculatum δ-Proteobacteria/Desulfovibrionales Desulfomicrobium baculatum δ-Proteobacteria/Desulfovibrionales Desulfomicrobium-related Uncultured environmental δ-Proteobacteria Firmicutes/Clostridium-related δ-Proteobacteria Desulfovibrionales/Desulfovibrio-related δ-Proteobacteria/Desulfosarcina-related δ-Proteobacteria/Desulfobacterium-related Verrucomicrobia-related Uncultured environmental δ-Proteobacteria Uncultured environmental δ-Proteobacteria Desulfosarcina-related/Desulfobacterales Desulfobacteraceae-related SRB Uncultured environmental δ-Proteobacteria Uncultured marine bacterium Uncultured environmental δ-Proteobacteria Desulfosarcina-related/Desulfobacterales Uncultured environmental δ-Proteobacteria Desulfosarcina-related/Desulfobacterales

5 1 1 1 4 1 3 1 2 1 5 4 1 1 3 2 2 1 2 1

13 6 4 3 16 3 5 9 6 1 17 20 2 13 7 5 10 10 19 4

93–97 99 99 85 94–95 96 91–96 91 93–94 91 90–98 98–99 98 98–99 98–99 93–99 97–98 97–99 98–99 99

Summer ANe layer

Winter AN layer

Summer SURFf sediment Summer LOWg sediment

Winter SURF sediment Winter LOW sediment a b c d e f g

Affiliation of 16s rRNA clones inferred from Fig. 4a, b. OTUs formed on the 97% threshold value. Homology determined from the NCBI database. Chemocline (8–9 m) water layer. Anoxic (12 m) water layer. Surface (0–5 cm) sediment. Lower (5–10 cm) sediment.

Fig. 4. Copy number of total bacterial 16S rRNA and 16S rRNA of detected SRB subgroups (DSV and DCC) in water (a) and sediment (b) layers sampled during the winter and summer season. Identical letters above the bars (mean ± standard deviation) indicate that differences were not significant according to the Kruskal-Wallis test (P b 0.05). Vertical scale bars represent standard deviation.

sediment layers. Diversity differences in SRB communities were confirmed by the rarefaction analysis. In contrast to findings in similar ecosystems of Lake Suigetsu and Lake Kaiike (Koizumi et al., 2004; Kondo and Butani, 2007), we observed that SRB community occupying water layer in Rogoznica Lake was more complex compared to the one found in the sediment probably reflecting more fluctuating conditions in the water layers than in the sediment. Specific SRB populations appearing either in the summer or winter sampling period indicated their seasonal dependence. In the summer water layers, in conditions of higher salinity, higher H2S concentration and total organic carbon (TOC), SRB community was less diverse then in the winter, probably due to community enrichment by highly adapted SRB subpopulation. Population dynamics in sediment, where diversity was higher during the summer could not be explained in the same manner, indicating that processes in the sediment are slower and less susceptible to the variation of physico-chemical and biological conditions, having prorogated response to the same changes in the water column. Several SRB populations showed unique vertical distribution, appearing specifically either in the certain water layer or at the specific sediment depth. An almost complete change in the composition of the SRB assemblage between the water and sediment as well as between specific seasons has also being recorded in meromictic lakes Kaiike and Suigetsu (Koizumi et al., 2004; Kondo and Butani, 2007). Rarefaction curves of the water layer samples (Fig. S2) were not fully saturated indicating that this zone of the Lake is much bigger reservoir of genetic variability then we expected. Based on the number of recovered clones the most abundant and the most diverse SRB in the water layer zone of the Lake were uncultured bacteria assigned to the δProteobacteria (populations S1, W2, Fig. 3a) with other populations fell within previously identified SRB: Desulfomicrobium, Desulfosarcina, Desulfobacterium and Desulfovibrio. In the anoxic water layer one unique population (S4) was detected in the summer, with no published sequence matching with N 90% similarity, therefore representing a novel lineage. Even though not affiliated to any known SRB, clear differences in the phylogeny of S1 and W2 δ-Proteobacteria-populations were observed between different sampling seasons. Similarly, Desulfomicrobium and Clostridium were found exclusive in the water layers of the Lake and in the summer season (populations S3, S5). SRB community switch was

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even more profound in the colder season when unique populations of Desulfosarcina, Desulfobacterium, Desulfovibrio and Verrucomicrobium appeared in the anoxic layer (populations W1, W3–5). Of these populations, Desulfovibrio-group represented the most diverse group (represented by 3 subpopulations) while Verrucomicrobia had only one representative. Detected SRB are commonly found in water column and sediments of freshwater, marine and brakish environments, such as Lake Pavin, Lake Sigetsu, Guayamas Basin, Colne River estuarine sediments, New England salt marsh (Kondo and Butani, 2007; Kondo et al., 2004; Lehours et al., 2005). Interestingly, Verrucomicrobia and Clostridium were likewise detected by using SRB-specific primer sets. Firmicutes, especially genus Clostridium as well as Verrucomicrobia are known to degrade polysaccharides, proteins and lipids and under fermentative process they can produce formate, acetate, lactate, and other low weight molecules that are specific nutritional substrates for SRB growth. It has been suggested by Martinez-Garcia et al. (2012) that few phylotypes of Verrucomicrobia, can make a considerable contribution to polysaccharide degradation, although they constituted only a minor fraction of the total microbial community. Since we did not identified bacteria belonging to the Syntrophobacteraceae family or other relatives, the oxidation of complex organic molecules in the Lake probably occurs via degrading activity of Clostridia and Verrucomicrobia which are likely triggering the activities of SRB populations. SRB were also detected in the chemocline, indicating that these bacteria occur even in the conditions of low oxygen concentrations. This community showed structure similarity to those found in the lower anoxic water layer. SRB were already detected in oxygenated and chemocline water layers, such as those of meromictic basins of the Black and Baltic seas (Bryukhanov et al., 2015). In both this and our study Desulfomicrobium emerged as a predominant phylogenetic group of SRB harboring chemocline layer. As it has been shown many of SRB species possess effective enzymatic systems of oxygen stresses protection and are able to form cell aggregates and symbiotic consortia with aerobic microorganisms. In addition, in oxic-anoxic interface of Rogoznica Lake, where highest POC concentration indicated organic particles accumulation, inner anaerobic microzones might represent optimal niches for SRB growth (Bryukhanov et al., 2015). Clone libraries of the sediment were found to be less complex than those in the water column. Majority of the retrieved sequences were unknown bacteria, affiliated to uncultured δ-Proteobacteria (population C1, Fig. 3b) with only one population correlated to the cultured SRB species Desulfosarcina (population C2). Surprisingly, none of the other SRB genera found in the overlaying water were present in the sediment. Compared to the water layer, Desulfosarcina-related SRB populations of Rogoznica Lake were present in both seasons throughout the sediment, but similarly, showed preferences toward winter conditions when more clones were retrieved. Members of Desulfosarcinales are usually dominant in marine environments (shallow arctic sediments, Eel River Basin and Santa Barbara Basin) comprising up to 73% of the total SRB (Orphan et al., 2002; Ravenschlag et al., 2000). Seasonal shifts in SRB community structure were also observed in the sediment. Populations of δProteobacteria were generally more abundant in the summer season. Four populations (correlated to uncultured δ-Proteobacteria and marine bacteria) were found exclusively in the summer and clearly correlated either with the surface (population S2, Fig. 3b) or lower (populations S1, S3, S4) sediment. Two populations (W1, W2) were designated as unique in the winter sediment. Phylogenetic affiliations of bacteria found in the winter sediment showed clear separation between the surface and lower SRB communities (data not shown) indicating depth distribution of different SRB already at this first few centimeters of the sediment. qPCR analysis showed that total bacterial density remained stable in the water layers and sediment over the sampled seasons, while SRB populations were shown to be more susceptible to seasonal changes. In general, SRB comprised only a small fraction of the total bacterial community. As expected, SRB community harboring sediment zone was more abundant then the one in the water layer comprising up to

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5% in summer surface sediment. SRB populations are usually found in low abundances in the anoxic water column where they account from 0.3 to 9% of the total bacterial community (Kondo et al., 2004; Neretin et al., 2007). In surface marine sediment SRB are usually more abundant, representing up to 26% of the community (Koizumi et al., 2004; Kondo et al., 2004; Leloup et al., 2006, 2004; Ravenschlag et al., 2000). Low abundances of SRB could be explained by the fact that microorganisms with a specialized metabolism often represent rare biosphere members fulfilling gatekeeper functions in an ecosystem while sustaining lowabundance populations, while those recognized as generalist are abundant in the bulk community (Inceoglu et al., 2015; Lynch and Neufeld, 2015). Most of SRB cannot use many organic compounds, especially complex polymers, and consequently an entire consortium is needed to mineralize organic material. However, qPCR suggested that SRB members of the DCC subgroup are more abundant in both water and sediments of the Lake compared to DSV subgroup. Reason for their higher abundance can be explained by the fact that DCC subgroup members are known as complete oxidizers that utilize a broader range of substrates. A number of studies have described seasonal changes of physicochemical parameters as the main factor shaping bacterial and phytoplankton communities (Ciglenečki et al., 2015; Kršinić et al., 2000; Pjevac et al., 2014; Gilbert et al., 2006, 2009; Fuhrman et al., 2006; Kan et al., 2007). For SRB, DOC concentration, type of the organic molecules, salinity and sulfate are parameters considered to play an important role in activity and structure of the community (Massana et al., 1997; Fortin et al., 2004; Kan et al., 2007; Eiler and Bertilsson, 2004; Fujii et al., 2012). During two sampling season measured parameters showed clear stratification of the water column. T and pH varied only slightly, whereas S changed for app. 5 psu in the anoxic layer and therefore could not be directly correlated to the observed community changes. Sulfide concentration was higher in the summer season, indicating higher activity of SRB. DOC and POC values follow the same pattern with higher concentrations during summer. In addition, DOC and POC values increased with the water depth with maximum found at the chemocline. Autochthonous DOC derived from primary production contains highly bioavailable substances, such as a variety of carbohydrates, amino acids, peptides and organic acids (Sundh, 1992). Accordingly, phytoplankton blooms have significant effect on the biomass and community structure of heterotrophic bacterioplankton, as previously reported (Eiler and Bertilsson, 2004; Fujii et al., 2012; Van der Gucht et al., 2001). It is therefore possible that the differences in TOC quantity and quality (e.g. bioavailability) are the factors affecting bacterioplankton community and subsequently could shape SRB community of the Lake. Correlation of the organic matter with the appearance and/or enrichment of the certain SRB populations cannot be discussed with certainty because majority of the retrieved clones were not phylogenetically closely related to any to-date isolated SRB strains. However, if the physiology of the retrieved clones is similar to the cultured strains it could be suggested that the complete oxidizers (DCC subgroup) in the water layers favor winter conditions.

5. Conclusions This study explored for the first time behaviour of specific SRB community in the euxinic seawater environment of Rogoznica Lake. Results gave new insight into the diversity, abundance and phylogeny of SRB suggesting that this ecosystem represents quite vivid area where seasonal variations in diversity and abundance of SRB communities in the water column and in the sediment were observed. Combination of various molecular methods enabled better characterization of SRB communities, while their abundance determined by qPCR varied slightly, sequencing showed notable changes in their community structure. Low sequence homology to cultured SRB indicated presence of the unique SRB community residing in the anoxic zone of the Lake.

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Rogoznica Lake represents a highly productive ecosystem hosting in a large scale uncharacterized sulfate reducers responsible for sulfidogenesis. Presented data show a complex SRB diversity and distribution supporting the idea that habitat-specific SRB communities are important part of the microbial consortium in the anaerobic degradation of organic matter as well as cycling of sulfur and carbon species in the Lake and similar anoxic environment. Acknowledgments This study was supported by Croatian Science Foundation through the project IP-11-2013-1205, SPHERE. We are grateful to Jelena Dautović for POC measurements Zdeslav Zovko for DOC measurements. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jmarsys.2017.03.003. References Baatar, B., Chiang, P.-W., Rogozin, D.Y., Wu, Y.-T., Tseng, C.-H., Yang, C.-Y., Chiu, H.-H., Oyuntsetseg, B., Degermendzhy, A.G., Tang, S.-L., 2016. 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