1. Introduction

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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
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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;
PO43-, up to 22 µM; SiO44-, up to 400 µM) [2], [6], as well as dissolved organic carbon (DOC up to 6 mgl-1),
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 TrisHCl, 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,
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.
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Rogoznica Lake – an extreme environment hosting specific sulfate-reducing bacterial community
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 layer above 6 m in both seasons, and more saline and denser bottom layer.
During both seasons CC layer was between 8-9 m, under which anoxic conditions were established.
Figure 2. Vertical profiles of temperature, salinity and oxygen in Rogoznica Lake measured during winter season 2014. (A)
and summer season 2014 (B)
B
A
0
10
20
30
40
0
2
2
4
4
Depth (m)
Depth (m)
0
6
8
10
Temperature (°C)
Salinity
Oxygen (mg/l)
0
10
6
8
10
12
12
14
14
Temperature (°C)
Salinity
Oxygen (mg/l)
20
30
40
4th International Conference on Research Frontiers in Chalcogen Cycle Science & Technology
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 DesulfococcusDesulfonema-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
name
Predicted target group
Water
(12m)
Sedim.
(0-5 cm)
Sedim.
(5-10 cm)
Water
(8-9 m)
Water
(12m)
Sedim.
(0-5 cm)
Sedim.
(5-10 cm)
16S
rRNA
DFM
Desulfotomaculum
-
-
-
-
-
-
-
16S
rRNA
DBB
Desulfobulbus
-
-
-
-
-
-
-
16S
rRNA
DBM
Desulfobacterium
-
-
-
-
-
-
-
16S
rRNA
DSB
Desulfobacter
-
-
-
-
-
-
-
16S
rRNA
DCC
Desulfonema
Desulfococcus
Desulfosarcina
/
/
+
+
+
-
-
+
+
16S
rRNA
DSV
Desulfovibrio
/
Desulfomicrobium
+
-
-
+
+
-
-
Target
gene
‘’+’’ 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.
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Rogoznica Lake – an extreme environment hosting specific sulfate-reducing bacterial community
CC layer summer
DSV winter AN water layer
DSV summer CC water layer
OTU1
OTU2
1000
DSV summer AN water layer
OTU3
OTU4
OTU5
OTU6
AN layer - winter
AN layer - summer
OTU1
OTU1
OTU2
OTU2
OTU3
OTU3
OTU4
OTU5
OTU4
OTU6
OTU5
OTU7
OTU6
OTU8
OTU7
OTU9
0.1
1000
1000
993
943
1000
845
Pseudomonasaeruginosa strain DSM 50071T
DSV 80
DSV 92
DSV 73
DSV KK12
DSV KK8
1000
Uncultured bacterium clone SPHAL 52 Synechococcus -related
Synecococcus sp KORDI-15
DSV 95
DSV 84
945
DSV 75
DSV KK22
DSV KK21
DSV 78
DSV KK9
DSV KK23
DSV KK13
DSV KK7
DSV AN36
DSV AN26
DSV AN34
Summer Cluster 1
DSV AN27
818 DSV KK24
Uncultured δDSV AN46
DSV AN48
Proteobacterium
DSV AN34DSV KK10
DSV
AN39
389
DSV AN28
Uncultured bacterium p763 b 4 41
Uncultured bacterium PM-150-Bact-09
106 999
Uncultured bacterium C9001C B156 C041
885
Uncultured bacterium methane seep Tainan Ridge
DSV 88
Uncultured bacterium Ld1 55 Nort Yellow sea sediments
Uncultured organism SBZO 1841 Guerrero Negro hypersaline
111
Uncultured_planctomycete_clone_OGT_B4_14
Planctomycete OGT B4 14 hadopelagic sediments
Uncultured environmental
Uncultured δ –Proteobacterium MothraB6-68
δ-Proteobacteria
DSV AN42
427
649 DSV KK14
911 DSV KK4
DSVAN40
DSV AN45
AN37
48
DSV
DSV AN30
DSV KK17
333
DSV KK11
Uncultured WS3 bacterium clone BFB005
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
DSVVerrucomicrobia
94
bacterium Sylt 22 Winter Sub-cluster Verrucomicrobium group
Thermodesulfovibrio yellowstonii Verrucomicrobia-related
DSV
AN31
437
148 1000
DSV AN48 1
Summer Sub-cluster 1a Uncultured environmental
DSV AN29
1000
Firmicutes
Clostridium sp BA-1
Clostridiales bacterium r28 Clostridium-related
1000
DSV 87
DSV 93
DSV 85
529 861
Winter
DSV 79
Uncultured Desulfobacteraceae ZLL-D59 Cluster 1
Uncultured environmental
639
Uncultured δ-Proteobacteria PET-124
DSV 91
448
δ-Proteobacteria
364
Uncultured
δDSV
89
282
DSV 82
566 SRB NaphS3
Proteobacteria Desulfobacterales
Uncultured δ-Proteobacterium YS-UMF1 C131
Uncultured bacterium JML C01
Uncultured δ -Proteobacterium_SGTA698
DSV 96
DSV 90
Winter
Desulfovibrio
hydrothermalis AM13
932
DSV
Cluster 2
DSV 8676
DSV 77
390
DesulfovibrioDesulfovibrio sp. An30N-mm
Uncultured clone CO1SHNF695
related
Desulfovibrio salexigens DSM 2638
Uncultured SRB 2R2V02
502 1000
δ-Proteobacteria
DSV AN33
DSVAN38
Desulfovibrionales
DSV AN35
DSVKK20
Summer
DSV KK19
DSV KK16
Cluster 2
DSV KK3
DSV KK2
Desulfomicrobium
DSV AN32
DSV KK2 1
baculatum
Desulfomicrobium baculatum HAQ-8
Desulfomicrobium baculatum
1000 Desulfomicrobium baculatum DSM 4028
Desulfomicrobium sp. PR4_C11
954
δ-Proteobacterium FM3-F10
995
DSV 74
Uncultured Desulfuromonadales bacterium clone B154 Winter Cluster 3 δ-Proteobacteria
Uncultured bacterium clone MD2894-B23
Desulfomonadales- Desulfomonadales
Desulfacter sp.
related
Uncultured Synechococcus_A82
Synechococcus sp. RS9918
Synechococcus sp._RS9918
Uncultured bacterium J608-77
Synechococcus sp. RCC36
Uncultured bacterium 014-12FFSCA001-A1B1 stratified seawater lake
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. δProteobacteria, 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 (9196% 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 δProteobacteria (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 (510 cm) samples grouped in 7 OTUs. All clones from SURF samples were related to uncultured environmental
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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
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.
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Rogoznica Lake – an extreme environment hosting specific sulfate-reducing bacterial community
Figure 7. Rarefraction analysis conducted with the Analytic Rarefaction software (http://www.uga.edu/strata/software/).
Δ DSV anoxic water layer winter
Χ DCC anoxic water layer winter
12
DSV anoxic water layer summer
DSV chemocline water layer summer
10
Number of OTU
8
6
4
DCC 0-5 cm sediment winter
ж DCC 5-10 cm sediment winter
▄ DCC 0-5 cm sediment summer
DCC 5-10 cm sediment summerr
2
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 almost
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
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 CO2.
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,
4th International Conference on Research Frontiers in Chalcogen Cycle Science & Technology
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 physicochemical 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.
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