ORIGINAL PAPER

Vertical physico-chemical gradients with distinct microbialcommunities in the hypersaline and heliothermal Lake Ursu(Sovata, Romania)

Istvan Mathe • Andrea K. Borsodi • Erika M. Toth • Tamas Felfoldi •

Laura Jurecska • Gergely Krett • Zsolt Kelemen • Erzsebet Elekes •

Katalin Barkacs • Karoly Marialigeti

Received: 22 July 2013 / Accepted: 25 January 2014 / Published online: 15 February 2014

� Springer Japan 2014

Abstract The effect of vertical physico-chemical strati-

fication on the planktonic microbial community composi-

tion of the deep, hypersaline and heliothermal Lake Ursu

(Sovata, Romania) was examined in this study. On site and

laboratory measurements were performed to determine the

physical and chemical variables of the lake water, and

culture-based and cultivation-independent techniques were

applied to identify the members of microbial communities.

The surface of the lake was characterized by a low salinity

water layer while the deepest region was extremely saline

(up to 300 g/L salinity). Many parameters (e.g. photosyn-

thetically active radiation, dissolved oxygen concentration,

pH, redox potential) changed dramatically from 2 to 4 m

below the water surface in conjunction with the increasing

salinity values. The water temperature reached a maximum

at this depth. At around 3 m depth, there was a water layer

with high (bacterio) chlorophyll content dominated by

Prosthecochloris vibrioformis, a phototrophic green sulfur

bacterium. Characteristic microbial communities with

various prokaryotic taxa were identified along the different

environmental parameters present in the different water

layers. Some of these bacteria were known to be hetero-

trophic and therefore may be involved in the decomposi-

tion of lake organic material (e.g. Halomonas, Idiomarina

and Pseudoalteromonas) while others in the transformation

of sulfur compounds (e.g. Prosthecochloris). Eukaryotic

microorganisms identified by molecular methods in the

lake water belonged to genera of green algae (Mantionella

and Picochlorum), and were restricted mainly to the upper

layers.

Keywords Lake Ursu � Hypersaline lake � Microbial

community � Stratification � Heliothermy � Green sulfur

bacteria

Introduction

Although saline lakes are globally distributed and con-

stitute 45 % of total inland waters (Wetzel 2001), and

there are increasing data available regarding the micro-

biology of these environments (Oren et al. 2009), still

little is known about the microbial diversity and ecology

of unique saline habitats, such as hypersaline heliother-

mal lakes. Halophilic and highly halotolerant microor-

ganisms can be found in each of the three domains of

life, Archaea, Bacteria and Eukarya, including photo-

trophic, chemoheterotrophic and chemoautotrophic

microorganisms (Cytryn et al. 2000; Oren 2002; Jiang

et al. 2006; Keresztes et al. 2012; Borsodi et al. 2013).

In the case of many saline lakes, the physical and

Communicated by A. Oren.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00792-014-0633-1) contains supplementarymaterial, which is available to authorized users.

I. Mathe � Z. Kelemen � E. Elekes

Department of Bioengineering, Sapientia Hungarian University

of Transylvania, Piata Libertatii 1, 530104 Miercurea Ciuc,

Romania

A. K. Borsodi � E. M. Toth � T. Felfoldi (&) � G. Krett �K. Marialigeti

Department of Microbiology, Eotvos Lorand University,

Pazmany Peter setany 1/C, Budapest 1117, Hungary

e-mail: tamas.felfoldi@gmail.com

L. Jurecska � K. Barkacs

Cooperative Research Centre for Environmental Sciences,

Eotvos Lorand University, Pazmany Peter setany 1/A,

Budapest 1117, Hungary

123

Extremophiles (2014) 18:501–514

DOI 10.1007/s00792-014-0633-1

chemical conditions change with depth and offer a great

variety of microhabitats for microorganisms (Cytryn

et al. 2000; Labrenz and Hirsch 2001; Glatz et al. 2006;

Demergasso et al. 2008; Keresztes et al. 2012).

Due to the intensive water heating process below the

slightly saline surface layer and the extremely high salt

content in the deeper zones, the hypersaline and helio-

thermal Lake Ursu (Romania) shows a specific thermal

profile. In fact a double stratification exists: (1) an

inverse thermal stratification with increasing temperature

up to the depth of 1.5–3.5 m (with maximum around

35–45 �C) and (2) a direct thermal stratification with

decreasing temperature from the thermal level to the

maximum depth. This double stratification appears over

spring and lasts until autumn (Alexe et al. 2006). Depth-

specific changes of water temperature and salinity define

a meromictic character to the lake, with a hypersaline

quasi-stagnant monimolimnion (= hypolimnion) and a

mixolimnion (= epilimnion) with lower salinity, supplied

by fresh and brackish water of the Toplita and Auriu

creeks (Bulgareanu et al. 1985). Since the end of the

19th century, the physico-chemical stratification, espe-

cially the thermal and saline stratification of Lake Ursu,

has been the subject of numerous studies (Kalecsinszky

1901; Maxim 1929; Bulgareanu et al. 1978, 1985; Alexe

et al. 2006, etc.). The first biological investigations

focused on the phytoplankton, the phytobenthos, the

macrophytic algae and the circumlacustrine cormophytic

flora which have an important contribution to the pelo-

idogenesis (mud forming processes; Bulgareanu et al.

1978). Later, these findings were supplemented with data

on the biological stratification of the lake: depth-specific

variation of the number of cultivable aerobic heterotro-

phic bacteria, density of Artemia salina, saprobity index

of the water and the structure of phytoplankton, zoo-

plankton and phytobenthos (Ionescu-Teculescu et al.

1982; Ionescu et al. 1998). Nevertheless, the species

composition of the bacterial communities has not yet

been examined in this lake, and such studies of helio-

thermal lakes are also sparse (Cytryn et al. 2000, La-

brenz and Hirsch 2001).

Therefore, the main objectives of the present study

were: (1) to explore the abundance and composition of

microbial communities inhabiting different depths for the

first time in this lake using cultivation-based (aerobic plate

count, bacterial strain isolation) and culture-independent

techniques (epifluorescence microscopy, denaturing gradi-

ent gel electrophoresis with DNA sequence analysis) and

(2) to reveal the possible relationships between the depth-

specific changes of the microbial communities and the

physical and chemical parameters in the stratified water

column of Lake Ursu during spring in two consecutive

months.

Materials and methods

Site description and sample collection

Lake Ursu is situated in Sovata (46�350N/25�040E) at the

foot of the Gurghiu Mountains (Mures County, Transyl-

vania Region, Central Romania) (Fig. 1). The hypersaline

meromictic Lake Ursu is one of the largest heliothermal

salt lakes in Europe (Bulgareanu et al. 1985; Muntean et al.

1999). The lake has a long axis of 366 m and a short axis of

239 m. The surface area is 41,270 m2 and the maximum

depth is 18.2 m (Alexe et al. 2006). The consequence of an

intense process of the Badenian (Middle Miocene) salt

massif dissolution, the lake has a karstosaline origin,

formed with sinking and the emergence of a big doline at

the confluence of the Toplita and Auriu creeks between

1870 and 1880 (Bulgareanu et al. 1985). The lake has a

10–15 cm deep surface layer of low salinity water

(15–54 g/L), resulting from rainfall and the two influent

creeks. Salt concentration increases up to the depth of

3–3.5 m where it can reach higher than 250–300 g/L val-

ues that remain constant until the depth of the sediment

surface. In the lake water, sodium and chloride ions dom-

inate while sulfate, hydrogen carbonate, calcium, magne-

sium and iron ions are present in lesser amounts

(Bulgareanu et al. 1985; Muntean et al. 1999; Alexe et al.

2006).

In this study, the water column above the deepest point

of the lake was chosen for sampling and in situ measure-

ments (Fig. 1). Water samples were taken from approxi-

mately 1, 3 and 9 m below the undisturbed water surface

on 28th March 2009 and from 0.5, 2, 3.5, 8 and 15.5 m on

24th April 2009 for analyzing the vertical stratification of

environmental variables. Water was pumped to the surface

using a 12 V diving pump with a flow rate of 1 L/min. The

Fig. 1 Geographical location of Sovata in Romania, and the position

of the sampling point in Lake Ursu (L lake, Cr creek). During summer

the lake is used as a health spa (area used by visitors appears striped)

502 Extremophiles (2014) 18:501–514

123

sampling tubes were rinsed with a tenfold volume of the

lake water before taking the sample. Samples for micro-

biological and physical and chemical analyses were trans-

ferred to the laboratory in a thermo box (at 5–10 �C) or

frozen on dry ice according to the parameter to be mea-

sured, and processing started within 24 h after sampling.

Aliquot samples were preserved on site for subsequent

chemical analyses: for total organic carbon (TOC) mea-

surements, samples were acidified with sulfuric acid

(0.01 M final concentration; MSZ EN 1484:1998); for

determination of sulfide ion concentration, 2 mL 5 %

Cd-acetate and 2 mL 2 M NaOH were added to the sam-

ples to the bottom of the sampling bottle, and immediately

closed without any air bubbles present. The solubility

product equilibrium constant (Ksp) of CdS is lower, than

the Ksp of ZnS, however, Cd(OH)2 has higher Ksp than

Zn(OH)2. Due to these facts cadmium salts are more

favorable for the separation of sulfide in alkaline environ-

ment as a precipitate, therefore Cd-acetate was used instead

of the conventionally applied Zn-acetate following the

guidelines of the Hungarian standard of wastewater ana-

lysis, which enables the detection of the sum of free

hydrogen sulfide and sulfides that could be released by

weak acids (MSZ 260-8:1968; Bethge 1954).

Determination of physical and chemical parameters

A DataSonde 4a meter (Hydrolab, Loveland, USA) was

used to measure in situ temperature (T), pH, redox potential

(ORP), dissolved oxygen (DO) and photosynthetically

active radiation (PAR) values from the water surface to the

depth of 15–16 m. A Turner Designs SCUFA on site

fluorometer was attached to the Hydrolab device to mea-

sure the combined concentration of ‘chlorophyll a ? bac-

teriochlorophyll c’ (Chl) of the water. Since it uses the

excitation wavelength 460 nm and emission wavelength

685 nm, and due to the similar fluorescent properties,

bacteriochlorophyll c could be also detected with this

equipment similar to chlorophyll a. The presence of both

pigments in Lake Ursu has been confirmed with in vivo

absorption spectrum analysis at a subsequent sampling

(Boglarka Somogyi, unpublished results).

Total bound nitrogen (TN) was determined by a Multi

N/C 2100S analyzer (Analytik Jena, Germany; MSZ EN

12260:2004) while salinity, hydrogen carbonate, reactive

phosphate, sulfate and sulfide ion, TOC concentration and

total iron content were determined according to Standard

Methods (Eaton et al. 2005) with some special consider-

ations as described in detail in Borsodi et al. (2013). In all

photometric detection, the salinity of the water was taken

into account, and adequate calibration curves were made

with salt-containing standards corresponding to the salinity

values measured at different depths of the lake.

Determination of total cell count

For the determination of total cell count (both live and

dead), a DAPI staining procedure was applied (Porter and

Feig 1980; Daims et al. 2005). Ten mL from each sample

was filtered through a 0.2 lm pore-size polycarbonate filter

(GTTP, Millipore, Billerica, USA), then filters were fixed

overnight with 2 % paraformaldehyde solution (direct fix-

ation of water samples was not possible due to the high salt

concentration of the water). Filters were washed with

1 9 phosphate buffered saline (8 mM Na2HPO4, 2 mM

NaH2PO4, 7.6 g/L NaCl, pH 7.2–7.4) solution and stained

with DAPI (40,6-diamidino-2-phenylindole; 1 lg/mL).

Total cell count values were determined with a Nikon80i

epifluorescence microscope using the Image-ProPlus 6.0

program package. For each sample, cell counts were

determined based on 25 different microscopic fields.

Determination of aerobic bacterial plate count

Aerobic heterotrophic plate counts were determined from

the tenfold serial dilution of water samples on sea water

agar (SWA) supplemented with 2, 7.5, 15 and 20 % NaCl,

Halomonas (HA), Marinococcus albus (MA) and R2A

Medium (R2A) supplemented with 15 % NaCl (Medium

246, 276, 434 and 830; http://www.dsmz.de). The pH of all

media was adjusted to 7.5. Colony forming units (CFUs)

were determined after 7–14 days of aerobic incubation at

25 �C by the visual observation of bacterial colonies.

Total genomic DNA extraction and denaturing gradient

gel electrophoresis (DGGE) analyses

For each water sample taken in March, an aliquot of

50–150 mL was filtered onto a 0.45 lm pore-size cellulose

nitrate filter (Sartorius, Gottingen, Germany) with gentle vac-

uum. Environmental genomic DNA was extracted from the

filters using the UltraCleanTM Water DNA Isolation Kit

(MoBio Laboratories, Carlsbad, USA) according to the man-

ufacturer’s instructions.

PCR amplifications were carried out using the primers and

cycling parameters listed in Table 1. Reactions were performed

in a final volume of 50 lL using 0.5–3 lL of genomic DNA,

0.2 mM of each deoxynucleotide, 2 mM (in the case of Euk-

arya-specific PCR, 1.5 mM) MgCl2, 2 U LC Taq DNA poly-

merase (Fermentas, Vilnius, Lithuania), 1 9 PCR buffer

(Fermentas), 0.4 lg/lL BSA (Fermentas) and 0.325 lM of

primers. In the case of Archaea, a semi-nested protocol was

applied (Table 1), a similar approach utilized in our previous

study of another saline lake (Borsodi et al. 2013). PCR pro-

ducts were visualized in an ethidium bromide-stained 1 %

agarose gel (SeaKem� LE Agarose, Cambrex Bioscience,

Rockland, USA) with UV transillumination.

Extremophiles (2014) 18:501–514 503

123

DGGE analysis with an INGENYphorU-2 electropho-

resis system (Ingeny International BV, Goes, The Nether-

lands) and the sequence analysis of selected bands on a

Model 310 Genetic Analyzer with a BigDye� Terminator v3.1

Cycle Sequencing Kit (Applied Biosystems, Foster City, CA,

USA) were performed as described in Felfoldi et al. (2009).

Identification of determined sequences was conducted with

database searches using the BLAST program (Altschul et al.

1997) and the GenBank nucleotide database while the deter-

mination of the closest type strain was carried out by EzTaxon

(Kim et al. 2012). The obtained nucleotide sequences were

submitted to GenBank under the following accession num-

bers: GU808778–GU808796.

Isolation and identification of bacterial strains

Bacterial strains were isolated from SWA and HA media.

From each growth medium, single colonies with different

morphologies were isolated on the same medium from the

highest dilution still showing growth.

Genomic DNA from the strains was extracted using the

Bacterial Genomic DNA Mini-prep Kit (V-gene Biotechnol-

ogy, Hangzhou, China) according to the manufacturer’s

instructions. The 16S rRNA gene was amplified by PCR with

the universal eubacterial primers 27F and 1492R (Table 1)

with a similar composition of reagents described above.

Strains were grouped based on the restriction patterns

(ARDRA—amplified ribosomal DNA restriction analysis)

generated by the endonucleases Hin6I and AluI (Fermentas)

which were applied in previous studies of saline aquatic eco-

systems (Felfoldi et al. 2009; Borsodi et al. 2010). Sequence

analysis of selected strains using primer 518R (Table 1) and

their identification with the EzTaxon tool and the GenBank

database were performed as described above. Nucleotide

sequences obtained were submitted to GenBank under the

following accession numbers: GU808797–GU808823.

The phylogenetic tree was constructed by the neighbor-

joining algorithm using Kimura’s two-parameter nucleo-

tide substitution model with the MEGA 4.0 software

(Tamura et al. 2007). Tree topology was re-examined by

the bootstrap method using 1,000 replications.

Results

Physical and chemical parameters

Previous studies on heliothermal lakes (e.g. Cohen et al.

1977a; Labrenz and Hirsch 2001; Alexe et al. 2006)

showed that stratification is a central limnological feature

of these lakes. We measured parameters directly related to

heliothermy, such as salinity and temperature, and other

physico-chemical characteristics (e.g. concentration of

nutrients) at different strata to better understand the envi-

ronmental background for the discussion of vertical

microbial distribution.

Table 1 PCR primer sequences and thermal profiles used in this

study for the selective amplification of the small subunit rRNA gene

from the major microbial groups of Lake Ursu

Primer

name

Primer sequence

(Reference)

Thermal profile

Strain

identification

27F 50-AGA GTT TGA

TCM TGG CTC

AG-30 (Lane

1991)

98 �C for 5 min, 32

cycles (94 �C for

30 s, 52 �C for

30 s, 72 �C for

1 min), 72 �C for

10 min1492R 50-TAC GGY TAC

CTT GTT ACG

ACT T-30 (Polz

and Cavanaugh

1998)

Archaea

DGGEaA109F 50-ACK GCT CAG

TAA CAC GT-30

(Baker et al.

2003)

98 �C for 3 min,

20 cycles (94 �C

for 30 s, 60 �C

for 30 s, decreas-

ing with 0.5 �C

in each cycle,

72 �C for 1 min),

15 cycles (94 �C

for 30 s, 50 �C

for 30 s, 72 �C

for 1 min), 72 �C

for 10 min

A340F 50-CCC TAC GGG

GYG CAS CAG-

30b (Baker et al.

2003)

A934R 50-GTG CTC CCC

CGC CAA TTC

CT-30 (Baker

et al. 2003)

Bacteria

DGGE A

27F 50-AGA GTT TGA

TCM TGG CTC

AG-30b (Lane

1991)

98 �C for 5 min,

32 cycles (94 �C

for 30 s, 52 �C

for 30 s, 72 �C

for 30 s), 72 �C

for 10 min518R 50-ATT ACC GCG

GCT GCT GG-30

(Muyzer et al.

1993)

Bacteria

DGGE B

968F 50-AAC GCG

AAG AAC CTT

AC-30b (Nubel

et al. 1996)

98 �C for 5 min,

32 cycles (94 �C

for 30 s, 52 �C

for 30 s, 72 �C

for 30 s), 72 �C

for 10 min1401R 50-CGG TGT GTA

CAA GAC CC-30

(Nubel et al.

1996)

Eukarya

DGGE

Euk1A 50-CTG GTT GAT

CCT GCC AG-30

(Dıez et al. 2001)

98 �C for 3 min,

32 cycles (94 �C

for 30 s, 56 �C

for 45 s, 72 �C

for 1 min), 72 �C

for 10 min

Euk516r 50-ACC AGA CTT

GCC CTC C-30c

(Dıez et al. 2001)

a A semi-nested PCR protocol was applied; the first PCR was con-

ducted with A109F-A934R and the second with A340F-A934R prim-

ers, in the latter case using the product of the first PCR as template DNAb A 39 bp GC-clamp was attached to the 50 end (50-CGC CCG CCG

CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG-30)c A 31 bp GC-clamp was attached to the 50 end (50-CGC CCG GGG

CGC GCC CCG GGC GGG GCG GGG G-30)

504 Extremophiles (2014) 18:501–514

123

In March, the water temperature reached its maximum at

about 5 m (24.8 �C) while in April the warmest tempera-

ture value (31.4 �C) was measured at 0.6 m (Fig. 2a). On

both sampling dates, a local temperature maximum was

recorded at around 3 m. Concentration of dissolved oxygen

and PAR decreased nearly to zero between 2.7 and 3.5 m.

Redox potential values decreased considerably at around

2–2.5 m from approximately ?200 mV to -150 mV.

Therefore, the lake water became anoxic and light-limited

at around 2.5–3 m depth. At the same depth, pH values also

shifted from slightly alkaline to slightly acidic. The Chl

concentration had an intensive maximum at 2.8 m.

Based on the results of chemical analyses of the water

samples (Fig. 2b), depth profiles of chemical variables could

be grouped into four distinct types: (1) profiles with a dramatic

change between 2 and 3.5 m but stable or slightly changing at

higher depths (salinity and TN); (2) a maximum value at

approximately 3.5 m (concentration of sulfide, phosphate and

hydrogen carbonate ions); (3) a continuous increase of the

values with increasing depth (sulfate ion and iron content); (4)

slightly changing values with increasing depth (TOC).

Enumeration of bacteria

In March, the highest aerobic CFU values were detected on

SWA supplemented with 7.5 % NaCl (1.5 9 104 CFU/mL

at 1 m, 1.2 9 103 CFU/mL at 3 m and 1.1 9 101 CFU/mL

at 9 m depths). Comparable result was found using SWA

Fig. 2 Depth distribution of in situ measured physico-chemical

variables (a) and chemical parameters determined in the laboratory

(b) in the Lake Ursu water column in March and April 2009. For

abbreviations, see ‘‘Materials and methods’’. Note that, Chl values

refer to the combined concentration of ‘chlorophyll

a ? bacteriochlorophyll c’ present in the water. In case of parameters

determined in situ (a), measurements were performed in 50 cm

intervals (in the metalimnion, 10 cm intervals), while in the case of

laboratory measurements (b), measurements were performed at the

sampling depths given in ‘‘Materials and methods’’

Extremophiles (2014) 18:501–514 505

123

with 2 % NaCl while other media were inherently more

selective. On the surface of the MA medium, no growth

was detected, except in the case of the 3 m sample. Simi-

larly, the HA medium gave no results for the sample taken

at 9 m (Fig. 3). In April, the aerobic colony count maxima

were detected on the surface of the HA medium and SWA

supplemented with 15 % NaCl (3.2 9 103 CFU/mL for

both, Fig. 3). In the upper water layers, similar CFU values

were found with all four tested media, and there was no

observable growth on the surface of the HA medium and

SWA with 20 % NaCl below 3.5 m. As expected, the

aerobic CFU values showed a steep decrease with depth

(excepting MA medium in March), since no aerobic bac-

teria would live in the anoxic stratum below 3.5 m depth.

Total cell counts were 3–5 magnitudes higher than the

plate counts at each examined depth of the lake (*106–107

cells/mL, Fig. 3). Decrease in total cell counts with depth

was not significant, rather the 3.5 m sample showed a

slight maximum in April.

DGGE analysis

Total microbial community composition was studied in the

case of samples taken in March. The DGGE profiles

showed significant differences among the samples and

indicated the presence of various taxa (Archaea and Bac-

teria) in all depths (Fig. 4). In the case of the Eukarya, a

multiple banding pattern was observed only in the sample

taken from 1 m. The efficiency of DNA isolation and

amplification was weaker in the case of samples with high

salinity (it was most pronounced in the case of the sample

from 9 m). Unfortunately, amplification efficiency could

not be enhanced with changes of the PCR parameters (e.g.

the amount of template DNA). Archaea-targeted PCRs

showed no depth-specific differences in DNA amplifica-

tion, fragments were retrieved from all samples in high

numbers, although in this case, a nested PCR protocol was

applied (Table 1). The highest number of distinct bands

was retrieved from the sample having the lowest salinity

(sample taken from 1 m depth) in all cases.

DNA from 19 different bands was successfully re-ampli-

fied, unambiguously sequenced and identified (Table 2). Ar-

chaea-related sequences belonged to three major taxonomic

groups of Euryarchaeota (Halobacteria, Archaeoglobi and

Thermococci), sharing low pairwise similarity values to

described genera (Halobacterium, Halorubrum, Halobellus,

Archaeoglobus and Palaeococcus). DGGE bands corre-

sponding to the Thermococci-related sequence were restricted

to deeper regions while bands of the other two major archaeal

groups were detected in all three depths (Fig. 4).

Sequences from eubacterial DGGE were related to five

prokaryotic taxonomic groups (Bacteroidetes, Chlorobi,

Clostridia, Gammaproteobacteria and Deltaproteobacteria)

and green algal chloroplasts (Table 2). In some cases, the

retrieved sequences could be assigned to specific genera or

even species, such as Pseudoalteromonas, Prosthecochl-

oris vibrioformis (formerly known as Chlorobium vibrio-

forme) or Mantionella squamata. Depth-specific

differences of the microbial community were most pro-

nounced in the case of Bacteria-targeted DGGE profiles

(Fig. 4). Each sample from the three depths possessed its

own characteristic eubacterial community (e.g. a Pros-

thecochloris-dominated community at 3 m) and common

bands for all samples were rarely observed.

Sequences retrieved from eukaryotes were related to

small chlorophyte algae (Picochlorum and Mantionella), to

Rotifera (Brachionus) and to Crustacea (brine shrimp,

Artemia) (Table 2). In the samples taken from the deeper

regions of the lake, only the band corresponding to the

picoeukaryotic alga Picochlorum atomus was detectable,

although that band in the same position was only very

dimly visible in the 1 m sample (Fig. 4).

Identification of bacterial strains

Altogether 80 bacterial strains were isolated from the water

samples taken in March (57 strains from 1 m, 20 from 3 m

Fig. 3 Comparison of colony count and cell count values at different

depth in Lake Ursu water column in March and April 2009. For the

abbreviation of culture media, see ‘‘Materials and methods’’ (NaCl

content of each media is shown in parentheses). DAPI, total cell

count after staining with DAPI

506 Extremophiles (2014) 18:501–514

123

and 3 from 9 m). The grouping of the strains resulted in 27

ARDRA representatives. Based on partial 16S rRNA gene

sequence comparisons, the majority of the strains belonged

to the Gammaproteobacteria (90 %) while other strains

were assigned to different genera of the Alphaproteobac-

teria and Firmicutes (4 and 6 %, respectively). Contrary to

strains of Gammaproteobacteria, which were isolated from

each studied depth and applied media, strains affiliated

with Alphaproteobacteria were only cultivated from the

sample at the depth of 1 m. Among the bacterial strains,

species belonging to genera Pseudoalteromonas, Idioma-

rina, Vibrio, Marinobacter, Halomonas, Thalassospira,

Roseovarius, Bacillus and Staphylococcus were identified

(Fig. 5). Strains with 98–99 % pairwise nucleotide

sequence similarities to species Halomonas arcis, H. ve-

nusta, H. alkaliantarctica and Idiomarina loihiensis were

found to be the most prevalent among the Lake Ursu iso-

lates. At the same time, single isolates with low (92–96 %)

sequence similarities to species Marinomonas ostreistagni,

Sulfitobacter pontiacus and Roseovarius tolerans may

represent novel bacterial taxa.

Discussion

Water stratification in Lake Ursu

The temperature profiles (Fig. 2a) of the upper 2–5 m of

the lake water refer to heliothermy, which is a result of the

stratification of water layers with different salinities and

solar radiation (a scientific explanation for the phenomenon

was first given by Kalecsinszky 1901). Heliothermy is

influenced by the thickness of the upper freshwater layer,

daily sunshine duration, water salinity below the depth of

2.5 m and air temperature (Bulgareanu et al. 1985; Wetzel

2001). The water level was 25–30 cm higher in March than

in April, due to the freshwater input from rainfalls and

melting snow, which justifies the deeper temperature

Fig. 4 DGGE profiles of DNA amplified with four different group-

specific PCRs from Lake Ursu samples taken in March 2009. The

applied concentration of denaturant (%) is shown in parentheses.

Arrowheads ([) indicate excised and re-amplified bands (for

sequence identification, see Table 2)

Extremophiles (2014) 18:501–514 507

123

maximum. In April, the warm weather rapidly increased

the water temperature close to the surface (monthly rainfall

decreased from 53.6 mm in March to 5.3 mm in April and

the number of sunshine hours increased from 114.7 to

290.3 h). The observed water temperature profile was in

accordance with former studies of Lake Ursu (Bulgareanu

et al. 1985; Alexe et al. 2006) and other heliothermal lakes

(Hammer 1986; Gibson et al. 1997; Cytryn et al. 2000;

Wetzel 2001).

Significant differences between March and April were

observable only in the case of temperature, vertical profiles

of other measured parameters showed similar trends

(Fig. 2). However, it was observable that the transition

zone around 3 m depth (where redox, DO, PAR, etc. values

changed considerably) was presented at slightly different

depths in the two studied months (Fig. 2), which could be

explained by the fluctuating water level of Lake Ursu due

to the seasonally different freshwater inputs (Istvan Mathe

and Karoly Marialigeti, unpublished data).

The changes in pH between 2 and 3 m coincided with

the decrease of dissolved oxygen level (Fig. 2a). This

could be explained by the fermentative decomposition of

organic material by the facultative aerobic and anaerobic

microorganisms in the anoxic environment, like Thermo-

cocci, Archaeoglobi, Halobacteria and Bacteroidetes.

Subsurface oxygen maximum with supersaturated DO

values up to 200 % was observed in both months at around

1 m depth, probably as a result of the activity of algal

populations (Wetzel 2001). Although detailed algological

study was not performed for our samples, a subsequent

study identified dense populations of cryptomonads and

pico-sized cyanobacteria and eukaryotic algae in the upper

3 m layer of Lake Ursu (Boglarka Somogyi and Lajos

Voros, unpublished results). Members of the latter group

(Picochlorum atomus) were detected by DGGE as a part of

eukaryotic plankton in our analysis.

The observed ‘chlorophyll a ? bacteriochlorophyll c’

(Chl) concentration maximum at about 2.8 m (Fig. 2a)

may be due to photosynthetic green sulfur bacteria, sup-

ported by the results of microbiological analyses (sequence

analysis and microscopy; Table 2, Supplementary Fig-

ure 1) and the strong decrease of the H2S content in this

water layer (Fig. 2b). The PAR values in this zone were

very low (Fig. 2a). These green sulfur bacteria use H2S as

electron donor, oxidizing it finally to sulfate. This was in

correlation with the increasing sulfate ion content in this

and in deeper water layers (Fig. 2b). Reports on similar

phenomena, i.e. extremely high abundance of green sulfur

bacteria in the transition zone of a meromictic saline lake,

are not so common. In the meromictic saline Lake Shunet

(South Siberia, Russia), total number of green sulfur bac-

teria reached almost 107 cells/mL at around 5 m depth

(Rogozin et al. 2010). Similar cell count values were

detected in the meromictic Lake Vilar and in the holo-

mictic Lake Ciso (Northeast Spain) in April by Casamayor

et al. (2000). In the heliothermal Solar Lake (Egypt), the

abundance of green sulfur bacteria was slightly lower, but

they formed a dense population (up to 2 9 106 cells/mL) in

Table 2 Identified sequences retrieved from excised bands of various

group-specific DGGEs (shown in Fig. 4) from Lake Ursu water

samples taken on 28th March 2009

Code (accession

number)

Closest speciesa [Major taxonomic

group]

Similarity

(%)

Archaea DGGE

A1 (GU808778) (Halorubrum litoreum)

[Halobacteria]

91.0

A2 (GU808779) (Halobellus limi) [Halobacteria] 92.0

A3 (GU808780) (Palaeococcus helgesonii)

[Thermococci]

81.3

A4 (GU808781) (Archaeoglobus fulgidus)

[Archaeoglobi]

81.0

A5 (GU808782) (Halobacterium noricense)

[Halobacteria]

92.9

Bacteria DGGE

B1 (GU808783) (Alkaliflexus imshenetskii)

[Bacteroidetes]

86.2

B2 (GU808784) Mantoniella squamata, chloroplast

[Chlorophyta]

98.4

B3 (GU808785) Pseudoalteromonas (marina)

[Gammaproteobacteria]

97.0

B4 (GU808786) (Desulfobacterium anilini)

[Deltaproteobacteria]

78.5

B5 (GU808787) (Desulfitobacterium

metallireducens) [Clostridia]

73.8

B6 (GU808788) (Alkaliflexus imshenetskii)

[Bacteroidetes]

81.5

B7 (GU808789) (Alkaliflexus imshenetskii)

[Bacteroidetes]

75.3

B8 (GU808790) Prosthecochloris vibrioformis

[Chlorobi]

99.5

B9 (GU808791) Prosthecochloris (vibrioformis)

[Chlorobi]

96.2

Eukarya DGGE

E1 (GU808792) Mantoniella squamata

[Chlorophyta]

99.6

E2 (GU808793) Brachionus plicatilis [Rotifera] 99.6

E3 (GU808794) Artemia franciscana [Crustacea] 99.4

E4 (GU808795) Artemia franciscana [Crustacea] 99.8

E5 (GU808796) Picochlorum atomus [Chlorophyta] 100

a In the case of prokaryotes, type strains based on EzTaxon search

while in the case of eukaryotes, closest species (excluding uncultured

sequences) based on Blast search are shown; distant relationships are

indicated with parentheses: in the case of prokaryotes [95 % pair-

wise nucleotide sequence similarity for genus and [97 % similarity

for species level were assumed as suggested by Tindall et al. (2010),

in the case of eukaryotes, no such general threshold values were

applied

508 Extremophiles (2014) 18:501–514

123

the upper hypolimnion at around 4 m depth (Cohen et al.

1977b) that corresponded with *90 lg/L Chl concentra-

tion. These values are in accordance to our results, since

the Chl peak of *150 lg/L around 3 m depth was asso-

ciated with *3–8 9 106 cells/mL of total microbial cells.

On the other hand, green algae could also contribute to the

high Chl fluorescent signal at around 2.8 m in Lake Ursu,

since they were the only phototrophic eukaryotes detected

by DGGE. However, to reveal the depth-shaped fine

structure of oxygen-producing small green algae and

anaerobic green sulfur bacteria, higher-resolution analysis

of stratification is needed in the future with more samples

collected from the same water column and with the

microscopic enumeration of these microorganisms.

Plate count of aerobic heterotrophic bacteria (*103

CFU/mL), as well as total cell counts (106–107 cells/mL) in

Lake Ursu was similar to those found in the heliothermal

and meromictic Ekho Lake in East Antarctica (Labrenz and

Hirsch 2001). However, it is interesting to note that the

number of cultivable aerobic bacteria detected on the same

SWA medium was at least one order of magnitude higher

in the neighboring Lake Rosu (Sovata, Romania; Fig. 1) in

Fig. 5 Neighbor-joining

phylogenetic tree based on the

16S rRNA gene sequence data

of strains isolated from the

water of Lake Ursu in March

2009. Bar represents 2 base

substitutions per 100 base pairs.

Thermodesulfobacterium

thermophilum was chosen as an

outgroup

Extremophiles (2014) 18:501–514 509

123

October 2003 (Borsodi et al. 2010). In Lake Ursu, the

decrease of aerobic CFU values as a function of depth can

be explained with the increasing salt concentration

(Fig. 2b), as well as a decrease in oxygen concentration

(Fig 2a). At the same time, total cell counts showed that the

amount of bacteria is rather high at each depth of the water

body, but most of these bacteria were not cultivable on the

applied media under the circumstances used. The DGGE

analysis clearly showed different populations of bacteria at

different depths (Fig. 4). The only parameter that showed

no dramatic change in the metalimnion was TOC

(Fig. 2b), and similarly to total cell counts, measured

values altered in a relatively narrow range (7.5–17.6 mg/

L, excluding the sampling point near the sediment sur-

face), since bacteria are not only important drivers of

organic matter degradation but also serve as a significant

source of TOC content in aquatic ecosystems (Wetzel

2001; Carstens et al. 2012).

Despite the high salt concentration in the lake water, a

diverse microbial community was detected, including

various genera of Archaea, Bacteria and Eukarya

(Table 2; Fig. 4). The DGGE results indicated significant

differences in the community patterns of the three

investigated depths (which was most pronounced in the

case of the Bacteria-specific DGGE), probably due to the

dissimilar physical and chemical environments (Fig. 2)

with a decreasing number of distinct DGGE bands with

increasing depth. Previous studies have shown that

increasing salinity may lead to the reduction of plank-

tonic microbial diversity at high salinities (e.g. Benlloch

et al. 2002; Casamayor et al. 2002). However, the rela-

tion of microbial diversity to lake water salinity is

ambiguous, since even the increasing influence of

recently accelerated diversification may contribute to

high diversity values at high salinities as suggested by

the DGGE-based study of Wang et al. (2011). In our

case, regarding Bacteria and Eukarya, the detected lower

band count in the more saline layers of Lake Ursu could

be affiliated with the low efficiency of PCR amplification

that was also indicated with low overall intensity of

DGGE bands (Fig. 4) (e.g. due to the lower abundance

of Bacteria and Eukarya in the deeper regions or the

high concentration of inorganic ions; von Wintzingerode

et al. 1997). In general, in our study, the whole diversity

of planktonic microorganisms was possibly not revealed

due to the limitations of the applied methods (e.g.

Amann et al. 1995, Casamayor et al. 2002, Nikolausz

et al. 2005), but we assume that main taxa were

identified.

Most of the measured physical and chemical parameters

showed minimal changes (salinity, water temperature) or

were constant (pH, ORP) below 5.5 m, indicating that Lake

Ursu was meromictic.

Taxonomic composition of planktonic microbial

communities in Lake Ursu

The phylogenetic analysis revealed that one-third of the

strains (32 %) were affiliated with different Halomonas

species (Fig. 5), some of which were described from

diverse saline and hypersaline aquatic environments on a

global scale; e.g. H. arcis from a salt lake on the Qinghai–

Tibet Plateau, China (Xu et al. 2007); H. janggokensis from

saline water in Anmyeondo, Korea (Kim et al. 2007); H.

alkaliantarctica from Lake Cape Russell, Antarctica (Poli

et al. 2007). The other most abundant cultivated group

(30 %) was identified as Idiomarina loihiensis, originally

described from a submarine hydrothermal vent (Donachie

et al. 2003). From all studied depths, strains belonging to

the genera Halomonas and Idiomarina were isolated using

media with various salt concentration. Our findings are in

accordance with the salt tolerance data of the type strains

of the closely related Halomonas and Idiomarina species

ranging between 0 and 20 % NaCl. The wide salt tolerance

of these genera could serve as a good foundation for

adaptation to the depth-changing salt concentrations pres-

ent in Lake Ursu.

Vibrio and Pseudoalteromonas species are typical

inhabitants of seawater, and they are frequently detected by

microscopy but are usually among the viable but non-cul-

tivable or difficult to cultivate bacteria (Du et al. 2007).

Only the presence of Pseudoalteromonas was detected by

cultivation and DGGE simultaneously, which could be the

consequence of the different selectivity associated with the

applied methods (e.g. Torsvik et al. 1990; Sipos et al.

2007), and fact that only a minority of the total bacterial

community could be revealed by cultivation (Amann et al.

1995). Therefore, isolated taxa potentially represent bac-

teria with low relative abundance.

One or just a few Lake Ursu strains represented the

genera Marinobacter, Thalassospira and Roseovarius or

were related to the genera Marinomonas and Sulfitobacter

(Fig. 5). All of their closely related halotolerant or halo-

philic type species were first isolated and described from

different saline aquatic environments (e.g. marine sedi-

ment, brine-seawater interface and hypersaline lake;

Sorokin 1995; Labrenz et al. 1999; Gorshkova et al. 2003;

Lau et al. 2006; Liu et al. 2007).

Members of ubiquitous Bacillus and Staphylococcus

species are able to tolerate higher salt concentrations, since

their survival is assisted by the synthesis of osmoprotec-

tants (e.g. choline, glycine betaine and proline), large cell

wall proteins and by changes of membrane phospholipid

composition (Oren 2008; Tsai et al. 2011). However, all of

the Lake Ursu strains affiliated with Firmicutes were iso-

lated only from medium SWA with lower salt concentra-

tion. Concerning the presence of a S. epidermidis-related

510 Extremophiles (2014) 18:501–514

123

isolate, it has to be emphasized that the lake is used as a

health spa in the warm summer period.

Borsodi et al. (2010, 2013) has studied the nearby saline

lake, Lake Rosu (Fig. 1), using some of the culture media

applied in this study, and recovered Halomonas, Marin-

obacter, Salinivibrio, Bacillus, Aurantimonas, Roseovari-

us, Psychrobacter, Serratia, Planococcus etc. isolates,

many of the genera detected also from Lake Ursu. Based

on a lower number of investigated strains (24 isolates), in a

recent study from a third saline lake in the same geographic

region (Lake Mierlei, Sovata; Crognale et al. 2013), similar

species have been identified (Halomonas, Marinobacter,

Salinivibrio, Bacillus, Idiomarina, Pseudoalteromonas,

and Staphylococcus). Both of the above-mentioned neigh-

boring lakes have similar chemical characteristics or even

connected by surface water flow (Lake Rosu) to Lake Ursu

and also used for bathing in the summer period, which can

explain the similarities in their bacterial community com-

position. However, contrary to Lake Ursu these lakes are

shallow (*1 m water depth).

Since the applied cultivation-based isolation protocol

allowed only the identification of aerobic heterotrophic

bacteria, molecular microbiological methods were also

used to survey the taxonomic composition of Lake Ursu

microbiological community. Among others, members of

the archaeal Halobacteria were detected in the lake, the

group that is well-known for inhabiting hypersaline envi-

ronments with a typical aerobic heterotrophic metabolism,

but many of them are able to grow anaerobically (Oren

2006). Considerable differences exist between the different

genera and species with respect to salt tolerance (Oren

2006), which was also indicated with depth-specific dis-

tribution of the Halobacteria-related phylotypes in Lake

Ursu. Thermococci, the other group of Archaea, was

restricted to deeper regions (most pronounced at 9 m), in

agreement with the obligate anaerobic nature of these

microorganisms (Bertoldo and Antranikian 2006). The

order Thermococcales is characterized by a fermentative

metabolism and for these organotrophic Archaea, in some

cases elemental sulfur is required for growth and used as

an electron acceptor being reduced to H2S (Bertoldo and

Antranikian 2006). The third group of detected Archaea is

also in connection with sulfur forms, since some Ar-

chaeoglobi are capable to obtain energy by reducing

oxidized sulfur compounds to H2S (Hartzell and Reed

2006) or to use sulfide, ferrous iron or H2 as electron

donors (Hafenbradl et al. 1996). The possibility of sulfate

reduction in the deeper regions of the lake was also

indicated by a distant relative of the well-known sulfate-

reducer Desulfobacterium among the retrieved DGGE

sequences from 9 m. The presence and activity of such

phenotypes are supported by the elevated sulfide levels in

these layers (Fig. 2b).

Phylotypes affiliated with the sulfur cycle of Lake Ursu

were also found among other eubacteria. The 3 m sample

was dominated with members of the genus Prosthecochl-

oris. These green sulfur bacteria could grow photo-

lithoautotrophically under anoxic conditions with reduced

sulfur compounds as electron donor (such as sulfide and

elemental sulfur), generating sulfate as the final oxidation

product (Imhoff 2003). During this oxidation process,

elemental sulfur is transiently deposited outside the cells

(Imhoff 2003) that might also be used by the Thermococ-

cales detected in the same layer. Prosthecochloris vibrio-

formis require at least 1 % NaCl concentration, the cells

are rods or vibroids occurring as single cells (Pfennig and

Overmann 2001; Imhoff 2003), which according to the

microscopic investigations, correspond with the charac-

teristic shape of cells in Lake Ursu at 3 m (Supplementary

Fig. 1).

Two photoautotrophic green algae were identified in

Lake Ursu with sequencing of the excised DGGE bands

(Table 2). Mantionella squamata (detected based on both

chloroplast 16S rRNA gene and genomic 18S rRNA gene)

was characterized by a cell diameter of 3–5 lm (Vaulot

et al. 2008) and was restricted to the upper water layer.

Picochlorum atomus was the only detected eukaryotic

organism in the 3 m layer of the lake and was almost

absent from the upper layer. These minute cells (2–3 lm)

of chlorophyte algae, lacking distinct morphological fea-

tures, were possibly identified as Chlorella vulgaris in

previous studies conducted with light microscopy on Lake

Ursu (Bulgareanu et al. 1978; Huss et al. 1999). Members

of the genus Picochlorum have broad halotolerance and are

inhabitants of various saline environments (Henley et al.

2002, 2004). In addition, these algae have been also

detected recently in some distant saline lakes of Transyl-

vania (Keresztes et al. 2012).

Two metazoan organisms, the brine shrimp Artemia

(that was also visible with the naked eye in the water) and

the rotifer genus Brachionus (Table 2) were detected with

community-based molecular biological investigation. Both

are possible predators of the bacterial and algal plankton,

although their presence was restricted to the upper aerobic

water layer in Lake Ursu.

Reports on the taxonomic composition of microbial

communities inhabiting heliothermal lakes are rather

scarce. Pioneering papers of Cohen and co-workers (Cohen

et al. 1977a, b, c; Jørgensen et al. 1979) gave a stupendous

and exhaustive description of the seasonal and vertical

changes of physical and chemical parameters and the

microbiology (based on mainly microscopic analyses,

radioactive labeling and cultivation) of the heliothermal

Solar Lake (Egypt), which has a completely different

hydrology than Lake Ursu, since Solar Lake is fed by

seawater seeping through a 60-m-wide gravel bar and has a

Extremophiles (2014) 18:501–514 511

123

short period of holomixis. Later, they have determined the

composition of archaeal communities in Solar Lake, and

found that halobacteria dominated with some uncultured

methanogens in the sulfide-rich deeper layers (Cytryn et al.

2000). However a detailed molecular diversity study of

Bacteria inhabiting different water layers of this lake is not

available to date. Labrenz and Hirsch (2001) has isolated

and described as new taxa various aerobic heterotrophic

bacteria from Ekho Lake in Antarctica, such as Fried-

manniella lacustris, Nocardioides aquaticus, Staleya gutt-

iformis, Antarctobacter heliothermus, Roseovarius tolerans

and Sulfitobacter brevis. Similarities between these and our

results could be found (e.g. the presence of halobacteria

within the archaeal community, some common heterotro-

phic bacterial genera, subsurface maximum of green sulfur

bacteria) due to the high salt content and stratified char-

acter of the water, while dissimilarities may be explained

partially with climatic and hydrological differences or the

methods applied. Finally, our study contributed to the

current knowledge regarding the depth-specific variation of

environmental parameters and the distribution of microbial

communities in different water layers of heliothermal

lakes.

Conclusion

The results of the examination of the hypersaline helio-

thermal Lake Ursu corroborated the importance of the

studies on extreme environments. The changes of physical

and chemical parameters with depth revealed the presence

of a characteristic metalimnion in the 2.5–3.5 m zone

marked by dramatic changes of redox potential, concen-

tration of dissolved oxygen, sulfide ion etc. In this layer

Chl concentration also reached its maximum due to the

dominance of the phototrophic sulfur bacterium Prosthec-

ochloris. At the same time, temperature changes did not

follow the same trend, but were influenced by the combi-

nation of factors as different salinity values of the water

layers and air temperature (i.e. thermal contact with the

atmosphere or intensity of solar radiation). Detected taxa of

Bacteria, Archaea and Eukarya showed a depth-shaped

distribution due to the increasing salinity and H2S levels

and decreasing dissolved oxygen and PAR values. Well-

known halophilic bacteria and phylospecies possibly rep-

resenting new taxa were similarly detected. Further studies

are needed to reveal the detailed role of limnological

parameters on the Chl maximum in the metalimnion.

Acknowledgments We thank to the Research Programs Institute of

Sapientia Foundation (Grant No. 209/37/2009) for supporting our

research and to grant POSCCE-A2-O2.1.1-2010-2 (No.

565/09.09.2013, Code: 1734, Acronym: SILOPREP) for providing

financial support for the establishment of Lake Ursu bacterial culture

collection. The authors wish to thank Rita Sipos and Eva Meszaros for

helpful discussions regarding the applied PCR-DGGE primers and

protocols. Meteorological data were provided by Gergely Makkai

from the National Meteorological Administration—Targu Mures

(Romania). We acknowledge the staff at Balneoclimaterica SA

Sovata, and we are also grateful to Toposervice SRL and to Laszlo

Szakacs for their help. T. F. was supported by the Janos Bolyai

Research Scholarship of the Hungarian Academy of Sciences.

Conflict of interest The authors declare that they have no conflict

of interest.

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