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Environmental Microbiology (2015) 17(10), 4105–4120 doi:10.1111/1462-2920.12979

Enhanced salinities, as a proxy of seawater

desalination discharges, impact coastal microbial
communities of the eastern Mediterranean Sea

Natalia Belkin,1 Eyal Rahav,2 Hila Elifantz,1 Introduction

Nurit Kress2 and Ilana Berman-Frank1*
1
Large-scale seawater desalination is an effective solu-
Mina and Everard Goodman Faculty of Life Sciences,
tion to the freshwater shortage of many countries around
Bar-Ilan University, Ramat Gan 52900, Israel.
2
the world. Desalination in the Mediterranean Sea com-
Israel Oceanographic and Limnological Research,
prises 17% of the world’s total desalination and is one of
National Institute of Oceanography, Haifa 31080, Israel.
three semi-enclosed basins with intensive desalination
activity that are anticipated to raise ambient salinities of
Summary the coastal habitats (Bashitialshaaer et al., 2011). In
Israel alone, seawater desalination by reverse osmosis
Seawater desalination plants increase local coastal
(SWRO) currently provides ∼450 million m3 (Mm3) y−1
salinities by discharging concentrated brine back to
of fresh water along the easternmost Mediterranean
the sea with ∼50% higher than ambient salinities. The
coastline. By 2025, water production supplied by five to
impacts of high salinities on microbial coastal popu-
seven large-scale coastal plants is forecast to reach
lations of the eastern Mediterranean Sea (EMS) were
750 Mm3 y−1 constituting ∼30% of Israel’s freshwater
examined in two mesocosm experiments; first, during
supply or c. 80% of the domestic and industrial needs
the mixed-spring and second, during the stratified-
(Dreizin et al., 2008).
summer periods with average salinity of ∼39. Ambient
The main by-product of all desalination processes is the
salinities were increased by 5% and 15%. Higher
large quantity of concentrated brine that is discharged
salinity (15%) mesocosms induced rapid (within 2 h)
back to the marine coastal environment (Ahmad and
declines in both primary productivity (PP) and algal
Baddour, 2014). With typical water recovery rates of
biomass parallel to an increase in bacterial produc-
40–50% in the desalination process, SWRO plants dis-
tivity. Subsequently, for the duration of the experi-
charge brine to the sea with nearly twice the salt concen-
ments (11–12 days), both Chlorophyll a and PP rates
tration of the ambient seawater. This discharge generally
increased (2 to 5 and 1.5 to 2.5–fold, respectively)
includes other chemicals used in the process (e.g. coagu-
relative to unamended controls. The initial assem-
lants, anti-foulants and anti-scalants) (NRC, 2008; UNEP,
blages of the ambient microbial populations and
2008; Spiritos and Lipchin, 2013). Along coastlines, water
intensity of salinity enrichments influenced the com-
loss due to its utilization by desalination plants, combined
munity responses. During the mixed-spring experi-
with brine discharge back to the coastal environment,
ment, the composition of prokaryotic and eukaryotic
increases the ambient salinity around the outfall areas
populations shifted only slightly, suggesting high
(Lattemann and Höpner, 2008). The temporal and spatial
functional plasticity of the initial populations.
dispersion pattern of the discharged brine differs among
While during the stratified-summer experiment, high
sites and seasons due to the discharge technology of the
salinity changed the composition and reduced the
plant, changes in local currents and annual physical–
biodiversity of the microbial communities. In an ultra-
chemical characteristics of the water column (Dawoud
oligotrophic environment such as the EMS, salinity
and Al Mulla, 2012).
induced declines in microbial diversity may provide a
The long-term environmental and ecological impacts of
tipping point destabilizing the local aquatic food web.
desalination plants on the marine ecosystem have been
poorly documented (Roberts et al., 2010). Yet, the
elevated salinity at discharge sites, frequently combined
with the chemicals applied during the desalination
process, may impact marine life and water quality as
Received 14 January, 2015; accepted 2 July, 2015. *For correspond-
ence. E-mail ilana.berman-frank@biu.ac.il; Tel. (+972)-3-5318214; changes in the physical and/or chemical environment are
Fax (+972)-4-6914842. often followed by shifts in the composition and production
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd
4106 N. Belkin et al.
Table 1. The initial physical, chemical and biological properties as measured on the first day of each of the two mesocosm experiments
(mixed-spring and stratified-summer experiments), 2 h after brine additions.

Mixed-spring experiment Stratified-summer experiment

Measured parameter Ambient 5% 15% Ambient 5% 15%

Salinity 38.8 ± 0.0 40.5 ± 0.1 40.5 ± 0.2 39.3 ± 0.2 41.3 ± 0.4 45.5 ± 0.5
Temperature (°C) 22.7 ± 0.0 22.7 ± 0.0 22.7 ± 0.0 29.9 ± 0.2 29.9 ± 0.1 30.0 ± 0.2
PO4 (μmol L−1) 0.13 ± 0.01 0.12 ± 0.02 0.18 ± 0.04 0.02 ± 0.00 0.02 ± 0.00 0.05 ± 0.01
NO2 + NO3 (μmol L−1) 0.39 ± 0.11 0.55 ± 0.21 0.50 ± 0.06 BDL 0.21 ± 0.12 0.60 ± 0.21
Si(OH)4 (μmol L−1) 1.57 ± 0.11 1.69 ± 0.11 1.79 ± 0.09 1.78 ± 0.08 1.86 ± 0.01 2.37 ± 0.04
Diversity of bacterial species 1762 ± 513 1656 ± 212 1729 ± 179 939 ± 322 1173 ± 212 1299 ± 122
Diversity of eukaryotic species 528 ± 138 480 ± 11 651 ± 31 173 ± 74 108 ± 57 131 ± 19
Chlorophyll a (μg L−1) 0.24 ± 0.02 0.12 ± 0.03 0.17 ± 0.01 0.24 ± 0.02 0.25 ± 0.02 0.17 ± 0.01
Primary productivity (μg C L−1 h−1) 3.36 ± 0.42 3.89 ± 0.58 1.77 ± 0.24 2.86 ± 0.39 2.74 ± 0.60 0.86 ± 0.15
PSII quantum yield (Fv/Fm) 0.12 ± 0.01 0.13 ± 0.02 0.07 ± 0.03 0.13 ± 0.00 0.11 ± 0.01 0.06 ± 0.02
Bacterial productivity (μg C L−1 h−1) 0.89 ± 0.61 2.52 ± 0.21 2.26 ± 0.30 2.34 ± 0.37 2.91 ± 0.69 3.26 ± 0.80
Bacterial abundance (105 cells ml−1) 6.3 ± 0.7 6.3 ± 0.3 5.7 ± 0.3 4.2 ± 1.1 4.3 ± 1.0 4.3 ± 0.5

All parameters are averages ± SD of three mesocosms per treatment.

BDL, below detection limit; Diversity, effective numbers of species that were calculated from Shannon Entropy Index.

of biological communities. Sensitive coastal environments
may especially be prone to alterations in salinity gradi-
ents, as observed for the low-salt tolerance Mediterra-
nean seagrass Posidonia oceanica and Cymodocea
nodosa (Sanchez-Lizaso et al., 2008; Garrote-Moreno
et al., 2014). Alterations in biodiversity and succession of
different species may also induce harmful cyanobacterial
or algal blooms further affecting coastal water quality
(Zingone and Enevoldsen, 2000).
Despite myriad studies discussing the potential for
adverse environmental impacts of desalination plant efflu-
ents, laboratory studies or field measurements assessing
these impacts are scarce (Roberts et al., 2010; Elimelech
and Phillip, 2011; Liu et al., 2013). Moreover, scant infor-
mation has been published showing the impacts of desali-
nation discharge on the autotrophic and heterotrophic
coastal communities (Drami et al., 2011; van der Merwe
et al., 2014). Yet, salinity is considered the most important
driver of global distribution patterns of bacteria as well as
other microorganisms (Lozupone and Knight, 2007) and
can regulate functional performance, growth rates
and shifts in bacterial community composition (Bouvier
and del Giorgio, 2002; Langenheder et al., 2003). Here,
we examined the impacts of increased salinity on the
structure and function of natural assemblages of plank-
tonic microbial populations from the coastal Eastern Medi-
terranean seawater (EMS) in 1 m3 mesocosms during two
11–12 day experiments. To simulate the natural salinity
increases near desalination plant outfalls along the Israeli
Mediterranean coastline (Roberts et al., 2010; Drami
et al., 2011; Kress et al., 2011), we enhanced salinities in
our experimental mesocosms by 5% and 15% above
ambient salinity (∼41 and 45 respectively). To account for
seasonal differences that influence the typical assem-
blages of the ambient microbial communities, we imple-
mented two identical experiments: one in early spring
when the water column is fully mixed and one in summer
when the water column is thermally stratified (termed
hereafter: mixed-spring experiment and stratified-summer
experiment respectively).
Results and discussion
Initial state and rapid physiological responses of the
microbial community to salinity
Following the salt additions, the water properties (tem-
perature and nutrients) retained the typical seasonal
values of the EMS surface waters (Table 1). Due to the
salt additions, inorganic nutrient concentrations were
slightly elevated but not significantly different (P > 0.05) in
the treated mesocosms relative to the control mesocosms
at the beginning of the experiment (T0) (elevations up to
0.05 μmol L−1 PO4, 0.5 μmol L−1 NO2 + NO3, 0.59 μmol L−1
Si(OH)4) (Table 1).
Seasonality imprinted the initial nutrient concentrations
and microbial assemblages of autotrophs and
heterotrophs, and these differences affected the subse-
quent microbial community responses. At T0, both bacte-
rial and eukaryotic representatives of the mixed-spring
experiment were more diverse than planktonic communi-
ties of the stratified-summer experiment (average effec-
tive number of bacterial and eukaryotic species 1715
versus 1137; and 553 versus 137 – during mixed-spring
and stratified-summer experiments respectively). In
summer, the bacterial community was a subset of the
spring community (Fig. S1) with a total overlap of 40.2 %
between bacterial operational taxonomic unit (OTU) from
the two seasons. A greater seasonal distinction was found
between the diatom and dinoflagellate populations com-
prising each experiment with only 13.4% and 16.2%

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4105–4120

respective identity in the composition of OTUs between
the mixed-spring community and stratified-summer com-
munity, with lower read numbers in the stratified-summer
experiment (Fig. S2).
The seasonal difference we detected in the OTU abun-
dance and composition of in-situ phytoplankton commu-
nities is consistent with published Mediterranean Sea
data demonstrating an increase in species diversity with
the annual enrichment of nutrients from winter mixing
(reviewed in Siokou-Frangou et al., 2010), which pro-
duces, along the Israeli coastline, a community comprised
of diatoms, dinoflagellates (micro-phytoplankton) and
nano-phytoplankton (3–20 μm) (Azov, 1986). In the
stratified-summer experiment, the phytoplankton diversity
was lower (Table 1), reflecting the typical summer plank-
tonic assemblages of the coastal Levantine waters domi-
nated by the small (< 3 μm) cyanobacteria and pico-
eukaryotic algae (Azov, 1986; Kimor et al., 1987; Herut
et al., 2014). Although diversity indices were higher for the
mixed-spring community than the stratified-summer com-
munity (Table 1), the initial algal biomass (Chl a) and
primary productivity (PP) were similar in our experimental
controls (ambient mesocosms) for both experiments
(0.24 μg Chl a L−1 and 3.15 μg C L−1 h−1, respectively,
and Table 1) and may actually reflect the coastal origins of
the waters that are more uniformly mixed, receive terres-
trial and anthropogenic nutrient inputs and depend to a
lesser extent than offshore populations on up-welled deep
nutrients.
In the EMS, heterotrophic bacteria contribute signifi-
cantly to the food web structure and may compete suc-
cessfully with phytoplankton when nutrients are limited
(Thingstad et al. 2005). Moreover, during thermal summer
stratification, a heterotrophic microbial food web sup-
ported by intensive recycling of organic carbon and nutri-
ents dominates the Levantine oligotrophic waters (Tanaka
et al., 2007; Ignatiades et al., 2009; reviewed by
Pulido-Villena et al., 2012). This was reflected in the
heterotrophic bacterial productivity (BP) that was
initially significantly higher in the stratified-summer experi-
ment than BP measured in the mixed-spring experiment
(2.3 μg C L−1 h−1 vs. 0.9 μg C L−1 h−1 respectively;
P < 0.005 – Table 1).
Regardless of the seasonal difference in the initial
diversity and composition, the salinity increases caused
an immediate functional response (within 2 h of salt
addition) followed by subsequent changes in both the
composition and function of the microbial communities
throughout the duration of the experiments. Moreover,
similar rapid physiological responses were detected in
both sets of experiments. Two hours after the brine addi-
tion, flow cytometry and molecular analyses revealed that
the microbial community structure remained unchanged
(Table 1, Table S1). Yet, rapid physiological responses
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4105–4120
High salinity impacts coastal microbial populations 4107
were recorded that were not a result of dilution. These
included suppression of photosynthesis and decreased
photochemical efficiency in the autotrophs and enhanced
heterotrophic bacterial activity.
Salt stress can inhibit various aspects of the photosyn-
thetic process (Sudhir and Murthy, 2004). Changes in
photosynthesis were reflected in the photosystem II
(PSII) photochemical efficiency (Fv/Fm) that decreased
significantly (P < 0.005) within 2 h in the 15% treatment
samples (Table 1). Salt stress can inactivate both photo-
synthetic reaction centres – PSI and PSII, and inhibit the
de novo synthesis of proteins, specifically the synthesis
of the D1 protein in PSII (Allakhverdiev et al., 2002). Salt
stress can also enhance the oxygenase activity of
ribulose 1,5-bisphosphate carboxylase/oxygenase while
curtailing its carboxylase activity and reducing carbon
fixation (Sivakumar et al., 2000). In cyanobacteria,
acclimation processes to salt stress can extend from
12 to 24 h, during which the cells activate osmolyte syn-
thesis (Hagemann, 2011). Our data suggest that the
addition of brine induced an immediate (within 2 h) salt
stress on autotrophic microorganisms and suppressed
photosynthesis.
Moreover, the photosynthetic pigment and indirect
proxy of algal-biomass (Chl a) decreased within the first
2 h (0.24 versus 0.17 μg Chl a L−1, in controls and in the
15% treatments, respectively, both in mixed-spring and
stratified-summer experiments) (Table 1). Changes in PP
paralleled those of Chl a. In the 15% mesocosms, PP
decreased significantly (P < 0.05) after 2 h from 3.4 to
1.7 μg C L−1 h−1 in the mixed-spring experiment and from
2.9 to 0.9 μg C L−1 h−1 in the stratified-summer experiment
(Table 1). The decrease in Chl a probably reflects the
immediate death of salinity-sensitive phytoplankton
species (Brand, 1984) following the salt addition. Moreo-
ver, salinity elevation reduces cellular Chl a content in
some algae and cyanobacteria (McLachlan, 1961) similar
to salt-susceptible plants. Yet, we could not find previous
published records of such rapid changes as we recorded
here.
Higher salinity also induced an immediate response
from the heterotrophic populations. Bacterial production
rates increased in the 15% treatments relative to controls
(by 2.5 and 1.5-fold in mixed-spring and stratified-summer
experiments respectively) (Table 1). Our results show
that under the experimental conditions, the abundance
of mixed-spring and stratified-summer coastal Mediterra-
nean bacterial communities was maintained at the
elevated salinities and was in the range of previous reports
in the EMS (Mapelli et al., 2013), while BP increased in the
higher salinity treatments (Table 1 – BA and BP ), indicating
higher cell specific bacterial activity. The rapid increase in
BP may be related to the elevated organic carbon con-
sumption required by bacteria to maintain the changing
4108 N. Belkin et al.

Fig. 1. Temporal changes in: A. Average Chl a concentrations normalized to controls; B. Carbon content of the ultra-phytoplankton community
at the outset and the end of the experiments (*denotes values are significantly different from other treatments P < 0.01); C. average primary
productivity (PP) normalized to controls. All averages include six biological replicates of mixed-spring and stratified-summer period
experiments combined ± SE; D. Average bacterial productivity (BP) normalized to controls, averages include three biological replicates of
mixed-spring and stratified-summer period experiments separately ± SE.

osmotic stress of cells and to regulate the internal pH as
well as changing the energetic potential of the cells’ mem-
branes (del Giorgio and Bouvier, 2002).
Physiological changes throughout the experiments
Salinity increases produced similar overall responses for
both experiments regardless of the season or initial micro-
bial inocula (Chl a; PP and ultra-phytoplankton biomass
composition) (Fig. 1). This similarity overshadowed some
microbial responses (e.g. BP) that were seasonally
dependent. During the course of each experiment, the
salinity remained unchanged from the initial values, while
temperature changed (up to 1.34°C) due to daily weather
fluctuations. At the end of the experiments (Tend), due to
biological utilization, inorganic nutrient concentrations
(PO4 and NO2 + NO3) were lower than at T0 and similar in
all mesocosms (Table S2). Si(OH)4 concentration was sig-
nificantly lower than T0 only in the 15% treatments indi-
cating changes in functional compositions (discussed
below).
In both experiments, the elevations in salinity resulted
in higher algal biomass (derived from Chl a concentration)
that was indicative of the adaptive capacity of the
autotrophic community. The 5% treatment caused a fast
and significant increase in the algal biomass after 2 days
(Fig. 1A) in both experiments, while autotrophs exposed
to 15% salinity lagged for several days before a significant
elevation in Chl a concentrations was recorded by day 5
(P < 0.001 and P < 0.005 for 5% and 15% respectively;
Fig. 1A). Subsequent increases in chlorophyll concentra-
tions to values ∼5-fold higher than control mesocosms
were measured at Tend (Fig. 1A).
The increase in autotrophic biomass was especially
pronounced in the ultra-phytoplankton which are impor-
tant primary producers in the Levantine basin (Azov,
1986; Siokou-Frangou et al., 2010) with small (< 5 μm,
pico-phytoplankton) cyanobacteria and larger (>5 μm,
nano-phytoplankton) eukaryotic algae dominant along
the Israeli coast (Herut et al., 2012). By Tend, ultra-
phytoplankton biomass increased significantly in the 15%
treatments (P < 0.01; Fig. 1B), contributed mostly by
larger (>5 μm) eukaryotic algal cell numbers (Table S1). In
the 15% higher salinity mesocosms these eukaryotic phy-
toplankton were probably diatoms as the stimulation of
algal biomass was accompanied by silica consumption.

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4105–4120

At Tend silica concentrations (in the 15% treatment
mesocosms) were 50–90% lower than T0 values and were
0.94 μmol L−1 and 0.11 μmol L−1 for the mixed-spring and
stratified-summer experiments respectively (Table S2).
The differential response in silica uptake is consistent with
the ability of diatoms to survive and thrive at the higher
salinity treatments (Kirst, 1989; Schapira et al., 2010) and
with the compositional changes of eukaryotes derived
from molecular analysis further described.
Concurrent with biomass changes, PP rates were 1.5-
fold higher in the 5% mesocosms throughout most of the
incubation period compared with T0 and parallel control
mesocosms (Fig. 1C). The most apparent and significant
change in PP rates was observed in the 15% mesocosms.
In these mesocosms, PP rates increased by 1.5 to fivefold
compared with control mesocosms 5 days after the T0
and were two to threefold higher than controls for the
next 7 days (P < 0.0005; Fig. 1C). In the first 5 days, PP
was significantly lower (by 0.7–0.8) than the controls
(P < 0.05), suggesting that the autotrophic community
function was initially inhibited by the salinity elevation
(Fig. 1C), similar to rates measured 2 h after experimental
induction (Table 1). These changes were likely caused by
osmotic stress that triggered species-specific metabolic
changes followed by structural changes of the community
during acclimation (Sudhir and Murthy, 2004; Kaartokallio
et al., 2005).
The observed lag time of 5 days prior to the enhanced
photosynthetic activity in the 15% treatments (Fig. 1C)
may have also been due to the higher energetic demand
of the phytoplanktonic community under elevated salt
conditions as reported from brackish water communities
(Pilkaityte et al., 2004). However, despite the enhanced
PP (after 5 days) in the stratified-summer experiment,
the PSII photochemical quantum yields (Fv/Fm) of the
autotrophic community were significantly lower in the
15% treatments than in the control mesocosms
(0.09 ± 0.03 versus 0.21 ± 0.07 respectively; P < 0.05).
PSII photochemical efficiency decreases when phyto-
plankton grow under stress, e.g. nutrient limitation or
high light (Kolber et al., 1988), indicating that salinity-
induced physiological stress caused the decline in Fv/Fm
in the 15% treatments. Similarly, exposure of the green
alga Chlorococcum cells to N-deficiency combined with
elevated salinities caused an inhibition of cell division
and a strong depression of photosynthetic activity
(Masojidek et al., 2000).
Excluding the rapid increases in BP measured during
the first hours of the experiments (discussed above),
salinity elevations did not significantly alter bacterial
heterotrophic production in most treatments. Seasonality
impacted the bacterial production potential as significant
elevation in bacterial production was measured only for
the mixed-spring experiment exposed to 15% treatments
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4105–4120
High salinity impacts coastal microbial populations 4109
from day 4 to Tend (P < 0.0005; Fig. 1D). No increase was
measured in BP for the stratified-summer experiment. The
contrasting response in the metabolic signature of bacte-
ria between seasons may have occurred due to the dif-
fering composition of the initial bacterial communities
(Fig. 2) and their functional plasticity.
Temporal changes in community composition
Compositional shifts within the different communities
derived from ribosomal ribonucleic acid (rRNA) genes
were examined using principal coordinate analysis
(PCoA). The distances (calculated from beta diversity)
between the beginning and the end (T0 and Tend) of the
experiment for each treatment indicate the extent of
the shift undergone by the communities (Table 2 and
Fig. 2). Throughout the mixed-spring experiment, the
composition of bacterial and eukaryotic groups was
similar for all treatments and for the control mesocosms
(Fig. 2A and B). These results indicate that salinity
increases barely affected the composition of these com-
munities and changes were primarily dictated by the
time of sampling.
In contrast, during the stratified-summer experiment,
salinity was a dominant driver impacting community com-
position as seen by the large change both in bacteria and
phototrophic eukaryote composition from the 15%
mesocosms (largest distances that significantly differed
from the controls; P < 0.005). After 6 days (Tm), both bac-
terial and eukaryotic communities in the 15% mesocosms
deviated from the control and 5% mesocosm (Fig. 2C and
D). Further analyses demonstrated shifts in both the
direction and magnitude of bacterial and eukaryotic alpha
diversity. This significantly changed from T0 to Tend
(Table 2). Thus, over the course of the experiment,
diversity of the ambient (controls) bacterial community
decreased by 51% while that of eukaryotes increased by
252% (Table 2). Concurrently, in the high-salinity (15%)
mesocosms, both bacterial and eukaryotic diversity
declined by 87% and 70% respectively (Table 2).
The three major phyla comprising 96–98% of all bacte-
rial OTUs from all stratified-summer mesocosms were
the Proteobacteria, Cyanobacteria and Bacteroidetes,
which are consistent with earlier reports from the EMS
(Feingersch et al., 2010). Significant shifts appeared
in the specific composition of proteobacteria and
cyanobacteria appeared during the experiment (12 days).
High salinity (15% addition) caused a 140% increase in
the relative abundance of cyanobacterial OTUs, while
cyanobacterial OTUs declined by 90% in the control
mesocosms (Table 2). In contrast, the relative abundance
of proteobacterial OTUs increased by 45% in the controls
and were negatively impacted by high salinity (15% addi-
tions caused a 30% decline).
4110 N. Belkin et al.

Fig. 2. Principal coordinate analysis based on beta diversity of species composition derived from analyses of bacterial 16S rRNA and
eukaryotic 18S rRNA genes in mixed-spring [A (16S) and B (18S)] and stratified-summer experiments [C (16S) and D (18S)]; The
communities exposed to 15% salinity elevation from the final day of the experiment of each experiment are marked by ellipsoids. T0: 2 h from
the beginning, T1: 1 day from the beginning, Tm: middle of the experiment, Tend: final day of the experiment.

The major cyanobacterial order in all mesocosms was

the Synechococcales (in particular Prochlorococcus Table 2. Temporal changes in microbial communities during the
stratified-summer experiment.
marinus), Oscillatoriophycideae and Nostocales. In the
15% treatment, P. marinus disappeared almost com- % Relative change Tend from T0
pletely after 1 day, while the abundance of filamentous
cyanobacterial OTUs, specifically Oscillatoriophycideae Salinity addition above control

and Nostocales, known to adapt to high-salinity conditions Treatment Control 5% 15%

(Hagemann, 2011; Jeffries et al., 2012), increased com-
16S True diversity −51% 82% −87%
pared with the controls (Fig. 3A, Fig. 4). Distance (median) 0.32 0.42* 0.59*
Among the heterotrophic bacterial groups, the Cyanobacteria OTUs −90% −45% 140%
alphaproteobacterium (Pelagibacteraceae) Pelagibacter Proteobacteria OTUs 45% 13% −30%
18S True diversity 252% 156% −70%
and the gammaproteobacteria Altermondales declined Distance (median) 0.33 0.39 0.60*
significantly after 6 days in the high-salinity (15%) Diatom algae OTUs −90% −86% 15%
mesocosms of the stratified-summer experiment, (Fig. 3B, Dinoflagellates OTUs 300% 400% 150%

Fig. 4). These were replaced by Rhodobacterales and
specifically Roseibacterium elongatum and Paracoccus
marinus of the Alphaproteobacteria (Fig. 3B), which are
aerobic, chemoheterotrophic, bacteriochlorophyll-
containing bacteria (Suzuki et al., 2006) and aerobic
bacteria producing the carotenoid adonixanthin
diglucoside (Khan et al., 2008). The disappearance of both
Pelagibacter (Pelagibacteraceae) and P. marinus from the
Relative changes are recorded from the start of the experiment (T0) to
the end (Tend = 11 days) and presented as % change from T0 of
true diversity for bacteria (16S) and eukaryotic (18S) species; calcu-
lated distances between communities are based on beta diversity;
and the relative changes (% change Tend from T0) in the relative
abundance of OTUs of the major representative taxa: cyanobacteria,
proteobacteria, diatoms and dinoflagellates.
*Significant community shift, relative to changes that occurred in
controls (Bonferonni corrected unifrac Monte Carlo significance test
P < 0.005).

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4105–4120

15% treatments is notable as Pelagibacter comprises
∼30% of the bacteria in the global oceans and dominates
the heterotrophic bacterial community composition (Morris
et al., 2002) including the EMS (Feingersch et al., 2010).
The cyanobacteria Prochlorococcus contribute globally
from 9% to 18% of net primary production, marine carbon
fixation and oxygen evolution (Partensky et al., 1999;
DuRand et al., 2001; Flombaum et al., 2013). Adaptations
to high-salt environments have been documented in the
Prochlorococcus genome, potentially encoding for
osmolyte production (Scanlan et al., 2009; Klahn et al.,
2010). The Pelagibacter genome encodes for transporters
of several osmolytes (Giovannoni et al., 2005) along with
the ability to metabolize these compounds (Sun et al.,
2011). Pelagibacter may actually consume osmolytes pro-
duced by Prochlorococcus and other phytoplankton as a
source of energy and nutrients (Thompson et al., 2013).
Thus, their sensitivity to high-salinity environments, such
as we observed here after 6 days (Tm), is intriguing and
suggests a profound shift in the planktonic food webs that
may occur near marine outfalls of desalination plants
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4105–4120
High salinity impacts coastal microbial populations 4111
Fig. 3. Temporal dynamics in the abundance
of major microbial lineages as obtained by the
relative OTU abundance ± SD (of total A, B:
bacterial, C, D: eukaryotic OTUs) throughout
the 11 days of the stratified-summer
experiment for the 15% enhanced-salinity
mesocosms. A: cyanobacteria; B:
proteobacteria; C: diatoms and D:
dinoflagellates.
during the stratified season of the eastern Mediterranean
(May–Dec).
During the stratified-summer experiment, dominant
autotrophic eukaryotic groups (diatoms and dinoflagel-
lates) changed concurrently with the altered bacterial
communities (Table 2). The initial populations of the
experiment were consistent with the coastal waters that
are relatively rich in the number of diatom species
(Gomez, 2003; Ignatiades et al., 2009; Herut et al., 2012).
In the 15% elevated salinity mesocosms, diatom OTUs
relative abundance increased by 15%, parallel to a
decrease of 86% to 90% in respective OTUs in the 5%
and control mesocosms. Here, the chain-forming diatom
Leptocylindrus spp. (50% to 60% of all diatom OTUs)
dominated the OTUs throughout the experimental period
(Fig. 3C) in contrast with the control mesocosms where
their relative OTU abundance declined from ∼50% at T0 to
0.2% at Tend (Fig. 5). Leptocylindrus spp. is the predomi-
nant diatom in some waters along the eastern and
western coastlines of the Mediterranean (Ignatiades
et al., 2009; Aktan, 2011; Herut et al., 2011; 2014). At the
4112 N. Belkin et al.

Fig. 4. Temporal changes in the composition of major bacterial lineages (Cyanobacteria and Proteobacteria) during the stratified-summer
experiment determined by the relative OTU abundance from 16S rRNA analyses at T0 and Tend – after 11 days for the control and 15%
enhanced-salinity mesocosms.

Fig. 5. Temporal changes in compositions of major eukaryotic lineages (diatoms and dinoflagellates) during the stratified-summer experiment
as determined by the relative OTU abundance from 18S rRNA gene analyses at T0 and Tend – after 11 days for the control and 15%
enhanced-salinity mesocosms.

© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4105–4120

15% salt mesocosms, Leptocylindrus was replaced by
increasing abundance of Minutocellus polymorphus (< 5%
at T0 to > 30% of eukaryotic OTUs after 8 days). This
pennate diatom is typical of enclosed and semi-enclosed
basins or estuarine waters, which may at times be heavily
eutrophied, and was previously found to bloom in a Medi-
terranean lagoon (Sarno et al., 1993).
Dinoflagellates comprised the second most abundant
group of eukaryotic phytoplankton in the summer experi-
ment with relative OTU abundances increasing by 300–
400% in the controls and 5% treatment communities and
by 150% in the 15% mesocosms from T0 to Tend (Table 2).
The two main genera identified in the 15% mesocosms
were Gymnodinium spp. (1% OTUs at T0) and Gyrodinium
spp. (1% OTUs at T0) (Fig. 3D, Fig. 5). Gyrodinium OTUs
increased to 12% of all eukaryotic OTUs by the end of the
experiment (Fig. 3D, Fig. 5) and dominated the dinoflagel-
late OTUs, while in control and 5% mesocosms their
relative abundance remained low (Fig. 5). Both these
genera are generally associated with warm and stratified
waters (Estrada, 1991), defined as truly phagotrophic, and
may constitute a main part of the microzooplankton (Sherr
and Sherr, 2007). Several Gymnodinium and Gyrodinium
species form red tides and harmful blooms and are
eurohaline (Zingone and Enevoldsen, 2000; Nagasoe
et al., 2006). Thus, their resiliency or increases under
high-salinity conditions should be of special concern when
monitoring outfall areas.
Our results present two types of responses of the micro-
bial community throughout the experiments. The microbial
community present during the mixed-spring experiment
went through minimal compositional changes yet
increased its metabolic activity in response to salinity
elevations. This suggests a high functional plasticity of an
initially resistant community, while the structural shift
during the stratified-summer experiment indicates a func-
tionally redundant microbial community that was controlled
by the salinity changes (Allison and Martiny, 2008). Biodi-
versity acts as a buffer against environmental fluctuations
and maintains the stability of ecosystem processes
(Tilman, 1999; Loreau et al., 2001). These principles
appear valid also for microbial systems (Bell et al., 2005;
Saikaly and Oerther, 2011). Higher diversity increases the
chance that some species will be resistant to changes,
allows species to compensate for one another and facili-
tates processes such as recruitment, thus enhancing
recovery over longer timescales. Maintaining the abun-
dance of species with an adaptive capacity, i.e. a combi-
nation of phenotypic plasticity, physiological responses,
distributional shifts and rapid evolution of traits better
suited to new conditions (Berga et al., 2012; Bernhardt and
Leslie, 2013) can stabilize community function (functional
redundancy) in a varying environment such as that with
altered salinity (McNaughton, 1977; Hooper et al., 2005).
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4105–4120
High salinity impacts coastal microbial populations 4113
In contrast, a negative correlation between salinity and
diversity may diminish the community resilience to addi-
tional disturbances such as increased temperatures
or extreme weather events associated with climate
change (Solan et al., 2004). Thus, decreases in bacterial
and phytoplanktonic diversities that can occur at the
outfall locations of desalination plants, and were also
exemplified in the high-salinity treatments of our stratified-
summer experiment (Figs 4 and 5), may reduce the com-
munities’ ability to overcome additional stressors.
Conclusions
Here, we demonstrated that salinity conditions similar to
those produced along the EMS coastline by desalination
brine disposal could be a driving factor shaping the
composition and function of the microbial planktonic com-
munities. We provide evidence for rapid physiological
responses (timescale of hours) that may occur even when
the residence time of plankton at the discharge site is
relatively short. Moreover, while brine discharges may
fluctuate temporally and spatially, their continual and long-
term (chronic) input to the coastal areas probably main-
tains the phytoplankton and bacterial communities under a
continuous state of salinity stress especially during periods
of low turbulence. Higher chronic salinity may reduce the
number of species and thus diversity, which would pre-
dominately select for high-salinity resilient organisms.
Adaptation to salinity fluctuations is crucial for planktonic
organisms and acts as a decisive factor regulating func-
tional properties of communities inhabiting a constantly
fluctuating system as found in coastal environments
(Brand, 1984). Thus, seasonal changes in composition and
flexibility in metabolic responses, as we measured, can
‘buffer’ against sudden environmental stress and increase
acclimation over longer periods of time.
Salinity-induced declines in biodiversity of primary and
bacterial producers in an ultra-oligotrophic environment
such as the Levantine basin may produce a tipping point
and destabilize the local aquatic food web including
grazers that may be directly impacted by higher salinities
(Hart et al., 1998). Furthermore, desalination outflows
also frequently discharge brine containing coagulants and
anti-scalants (i.e. iron salts and polyphosphonates,
respectively) that could further modify the responses of
planktonic communities to the altered salinity gradients
and should also be examined.
Experimental procedures
Mesocosm set-up
Acid washed polyethylene bags (nine bags, each ∼1 m3) sup-
ported by cylindrical plastic frames were deployed within a
continuously circulating seawater 16 m3 pool to maintain
4114 N. Belkin et al.
ambient temperature and illumination. Surface coastal sea-
water was pumped to these mesocosm bags from Tel
Shikmona (Haifa, Israel) at 2 m depth, approximately 300 m
from the coastline (32°49′34N, 34°57′20E). Bags were filled
with water using plastic tubing by alternating the filling among
the bags every 2 min to achieve a homogenous distribution.
The entire filling procedure lasted for ∼3 h. Polyethylene
covers prevented evaporation or dilution as well as external
contamination (atmospheric inputs, etc.), yet maintained gas
exchange with the atmosphere and allowed for ∼50% of
surface light to penetrate the water. The mesocosms were
located at the National Institute of Oceanography of the Israel
Oceanographic and Limnological Research IOLR in Haifa,
Israel.
Experimental design and sampling
Coastal mixed-spring shallow waters are expected to be
affected by the seasonal mixing processes of the open
waters, reflected in mixed-spring and stratified-summer
periods, when oligotrophic and ultra-oligotrophic conditions
prevail as well as different microbial communities (Krom
et al., 1991). These conditions influence the initial inocula
of the ambient microbial planktonic communities. Two
mesocosm experiments were carried out: (i) 23 April–5 May
2013 and (ii) 7–18 July 2013. These experiments were char-
acterized as mixed-spring and stratified-summer experiment
respectively. Both experiments consisted of a control
(ambient water – no salinity amendment) and two salinity
treatments (5% and 15% above ambient salinity), simulating
relative salinity elevations found at nearby desalination
outfall sites (Roberts et al., 2010; Kress et al., 2011), each in
triplicate mesocosms for statistical analyses. The 5% and
15% above ambient salinity (39.05 ± 0.35) represented an
average salinity of 40.90 and 45.25, respectively, in the
mesocosms. The salinity elevations were achieved by addi-
tion of artificial brine (80) prepared by dilution of seawater
salts (NēoMarine, Brightwell Aquatics) in ambient seawater.
The salinity was measured with a Yellow Spring Instruments
YSI 6000 probe (using the Practical Salinity Scale). To
prevent experimental bias due to dilution of the microbial
populations in the seawater, we prepared the salt additions
with the same seawater used in the mesocosms so that all
treatments and controls contained the same initial microbial
assemblage.
The nine mesocosms were filled and sampled 2 h after the
brine addition to fully characterize the initial properties of the
water and the community composition and function (time 0 –
T0). At each sampling, the polyethylene covers were opened
and 5–10 L water was collected gravitationally from each
mesocosm using acid washed Masterflex Tygon tubing into
acid washed 5 L plastic containers. Subsequent samplings
took place every 1–2 days at 08:00 am for a period of 11–12
days for salinity, temperature, chlorophyll (Chl a), bacterial
and primary productivity. Inorganic nutrient concentrations,
bacterial and ultra-phytoplankton abundance were measured
at the beginning and end of the mesocosm experiments.
Deoxyribonucleic acid (DNA) samples for bacterial and
eukaryotic composition were sampled on four occasions
throughout the experiment: days 0, 1, 6 or 7 (middle) and 11
or 12 (end), referred as: T0, T1, Tm and Tend respectively. To
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4105–4120
minimize biases due to sampling frequency causing a reduc-
tion of water levels, the water level within the mesocosms
was retained above 90% of the total volume throughout the
whole experiment (i.e. less than 100 L were taken out of
1000 L). To detect the community responses caused only
by the treatments, we compared all our results to the
unamended controls (incubated under the same conditions),
reducing the changes that occurred due to the enclosure of
the community or natural succession (Calvo-Díaz et al.,
2011).
Laboratory analyses
Seawater for inorganic nutrient analyses (ortho-phosphate,
nitrate + nitrite, nitrite and silicic acid) was sampled into acid-
washed scintillation vials. To prevent microbial decomposition
of organic matter, the samples were immediately frozen and
kept frozen until the day of analysis when they were thawed.
Nutrient concentrations were determined using a segmented
flow, Seal Analytical AA-3 System following the methods
described in Kress and Herut (2001). Quality assurance of
the methods was confirmed by the results of intercomparison
exercises (National Oceanic and Atmospheric Administration
(NOAA), USA and the National Research Council of
Canada (NRC), Japan, Quasimeme). The precision of the
nitrate + nitrite and nitrite, orthophosphate and silicic acid
measurements were 0.02, 0.003 and 0.06 μM, respectively,
while the limits of detection were 0.08 μM, 0.008 μM and
0.03 μM respectively.
Chl a concentrations were determined by the non-
acidification method (Welschmeyer, 1994). Mesocosm
samples (250 ml) were vacuum filtered through GF/F 25 mm
filters (Whatman) with a nominal pore size of 0.7 μm. The
pigments were extracted from the filters in 5 ml of 90%
acetone, at 4°C, for 24 h in the dark. Chlorophyll a concen-
tration was determined using a Luminescence Spectrometer
(Trilogy Laboratory Fluorometer, CA) at 436 nm excitation
filter, 680 nm emission filter.
Photosynthetic carbon fixation rates were measured by
a modified 14C incorporation method (Steemann-Nielsen,
1952). For each mesocosm, we filled quadruplicate
polycarbonate bottles (50 ml; Nalgene) with water during
morning (∼09:00), inoculated with 5 μCi of NaH14CO3 tracer
(Amersham, CFA3), and incubated for 4 h under ambient
irradiance and temperature (20.5–23.5°C and 28.5–30°C for
spring and summer experiments respectively). After incuba-
tion, particulate matter was collected on GF/F filter. The total
radioactivity in this fraction was determined by liquid scintil-
lation (Packard Tri carb 2100 TR liquid scintillation analyser)
and converted to rates of PP as described by Lagaria and
colleagues (2011) taking into account dark incorporation and
zero time controls.
PSII photochemical quantum yields (Fv/Fm) were deter-
mined using a Fluorescence Induction and Relaxation
System (FIRe – Satlantic, Halifax, Canada) to analyse the
photosynthetic characteristics of the autotrophic microorgan-
isms (Kolber et al., 1998). After 15 min of dark acclimation,
the samples were analysed with a FIRe system set to deliver
saturation flash sequences of 100 ms−1 with 1 ms−1 intervals
between flashes with the maximum gain (2400) utilized for all
samples. Fluorescence parameters measured were as

follows: Fluorescence F0 intrinsic fluorescence [arbitrary units
(a.u.)], the maximum fluorescence (Fm), when all PSII reac-
tion centres are photochemically reduced. Based on these
parameters variable fluorescence [Fv = Fm – F0 (a.u.)] was
determined. Fv/Fm was calculated after blanks (0.2 um filtered
seawater) were subtracted from the initial, dark, adapted
samples (Cullen and Davis, 2003).
Bacterial production rates were measured using the 3H
leucine incorporation technique (Kirchman et al., 1985).
Briefly, 1.7 ml triplicate samples and a control were incubated
with a mixture of L-[4,5-3H] leucine (Perkin Elmer, specific
activity 160 Ci mmol−1) at a final concentration of 100 nmol
Leucine L−1. Samples were incubated in the dark at room
temperature (∼25 °C), fixed and treated following the micro-
centrifugation protocol (Smith and Azam, 1992). Potential
bacterial production rates were calculated using a conversion
factor of 1.5 kg C mol–1 with an isotope dilution factor of 2.0
(Simon and Azam, 1989).
Ultra-phytoplankton and bacterial abundances were enu-
merated by flow cytometry. Samples of 1.8 ml were immedi-
ately fixed after sampling with 5 μl of 50% glutaraldehyde
(Sigma G-7651), incubated at room temperature for 10 min,
subsequently frozen in liquid nitrogen and kept at −80°C until
analysis. Prior to the analysis, fixed samples were fast
thawed at 37°C. Analysis was performed using a flow
cytometer – FACScan Attune, fitted with argon lasers (405
and 488 nm). Beads of 1 μm diameter (Polysciences) served
as standards (Marie et al., 2005; Stambler, 2006). The taxo-
nomic discrimination was based on cell side scatter – a proxy
of cell volume; forward scatter – a proxy of cell size; and
orange and red fluorescence of phycoerythrin and Chl a
(filters: 574/26 band pass and 640 long pass respectively).
Heterotrophic bacteria were stained (300 μl of the initial
sample) with SYTO 9 Green Fluorescent Nucleic Acid Stain
(Marie et al., 1997) and enumerated by discrimination based
on green fluorescence (530/30 band pass filter) and side
scatter.
Pico/nano phytoplankton carbon biomass was calculated
from cell counts assuming 175 fg C cell−1 for Synechococcus
cells, 53 fg C cell−1 for Prochlorochococcus cells and
2100 fg C cell−1 for nanoeukaryotes (Campbell, 2001).
Statistical analyses
Prior to the statistical comparison, data were binned into two
groups: T0–T5 and T6–Tend, to reduce the effects of minor
observation errors. To assess treatment-dependent signifi-
cant changes for each physiological parameter one-way
analyses of variance (ANOVAs) with permutations were per-
formed for each group using the R software package
(lmPerm, ver. 2.15). The permutation test allowed applying
ANOVA with no assumptions about the data set normality or
homogeneity of variance and corrected for the multiple com-
parisons. For comparison of each parameter between differ-
ent treatments, statistical analysis was done by post-hoc
Tukey honestly significant difference test after ANOVA test.
DNA isolation and high-throughput
phylogenetic analyses
To analyse the microbial community diversity by 16S and 18S
rRNA genes, water samples (2 L) were filtered on 47 mm
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4105–4120
High salinity impacts coastal microbial populations 4115
0.2 μm pore size Supor filters (Pall Gelman, Ann Arbor, MI),
frozen in liquid nitrogen and kept at −80°C until DNA extrac-
tion. Community genomic nucleic acids were isolated from
the filters, and media using a phenol–chloroform extraction
method modified according to Massana and colleagues
(1997) and Brinkhoff and Muyzer (1997). For initial amplifica-
tion, the broadly conserved bacterial primers 27F and 1100R
were used to amplify the 16S rRNA gene region (Lane, 1991;
Dowd et al., 2008), and eukaryotic primers 360F and 1492R
for 18S rRNA gene region (Edgcomb et al., 2011). Thermo
Scientific Phusion high-fidelity DNA polymerase was used to
amplify these segments. A secondary polymerase chain reac-
tion (PCR) was performed for next-generation sequencing
(Ion Torrent Life Technologies, USA) using specially designed
fusion primers with different tag sequences as: LinkerA-Tags-
27F and LinkerB-338R for 16S (Hamady et al., 2008);
LinkerA-Tags-528F and LinkerB-706R for 18S rRNA gene
(Cheung et al., 2010). Polymerase chain reactions were per-
formed under the following conditions: 95°C for 3 min fol-
lowed by 25 cycles and 20 cycles (first and secondary PCR,
respectively) of 95°C for 30 s; 60°C for 30 s and 72°C for
1 min and a final elongation step at 72°C for 5 min. After
secondary PCR, all amplicon products were purified using
Agencourt Ampure magnetic purification beads (Agencourt
Bioscience Corporation, MA, USA) to exclude primer dimers.
Products of DNA for the 16S and 18S fractions were
sequenced to get a representative view of the bacterial and
eukaryotic community composition.
Sequence analyses. Sequences were processed and ana-
lysed using ‘quantitative insights into microbial ecology’ (QIIME)
an open-source software pipeline (Caporaso et al., 2010).
Sequences were removed if they were < 200 or > 400 bp, had
a quality score of < 25, contained ambiguous characters or an
uncorrectable bar code or did not contain the primer
sequence. Remaining sequences were assigned to samples
by examining the bar codes. Mean number of sequences per
sample passing quality filters were 2823 and 5919 with
average sequence length 265 bp and 255 bp for bacteria and
eukaryotes respectively. Similar sequences were clustered
into OTUs using UCLUST (Edgar, 2010) with a minimum cov-
erage of 99% and a minimum identity of 97%. A representative
sequence was chosen from each OTU then was aligned using
PYNAST (Caporaso et al., 2010), the Greengenes (DeSantis
et al., 2006) and Silva databases (bacteria and eukaryota,
respectively) with a minimum identity of 80%. Chimera check-
ing was applied to remove chimera sequences, followed by
Lane mask to screen out hypervariable regions after align-
ment. Taxonomy was assigned using the Ribosomal Database
Project (RDP) [Michigan State University] classifier with a
minimum support threshold of 80% (Wang et al., 2007) and the
RDP taxonomic nomenclature.
Quantifying and comparing diversity. Several metrics were
applied to test the communities’ compositional shifts in the
mesocosms. All parameters tested were compared with the
control communities. To evaluate the diversity within commu-
nities (Alpha diversity), we employed rarefaction plots and
branch length-based phylogenetic diversity measurements
(Faith, 1992). Effective number of species (referred to as
diversity) were calculated from Shannon index of entropy (H)
4116 N. Belkin et al.
according to Jost (2007) using the conversion equation:
exp(H), to examine and compare the diversity of each com-
munity from different time points during the incubation period
(Moreno and Rodríguez, 2011). To determine the amount of
diversity shared between two communities (pairwise sample
dissimilarity – beta diversity), we employed the weighted
UNIFRAC metric (Lozupone and Knight, 2005; Lozupone and
Knight, 2007; Lozupone et al., 2007; Lozupone et al., 2011).
To control for sampling effort in beta diversity measurements,
rarefaction and jackknifing analysis were applied (Lozupone
et al., 2006). We performed significance tests and PCoA
using UNIFRAC (Lozupone and Knight, 2005). To further
understand the significant shifts, we calculated relative
changes in diversity of each treatment normalized to the
initial diversities. In addition, we calculated the extent of these
compositional shifts by testing the distances measured by
beta diversity between the beginning and the end of the
incubation for each treatment and tested which of the shifts
was significantly different from others (when P < 0.05) using
the UNIFRAC Monte Carlo significance test. Finally, we exam-
ined the OTUs of major representative taxa that changed
their abundance significantly (when P < 0.05) throughout the
incubation using Bonferroni corrected ANOVA and calculated
these relative changes.
Acknowledgements
We acknowledge the Israel Water Authority grant number
4500445459 for partial funding to Ilana Berman-Frank (IBF).
We thank Dan Miller for technical help and samplings
during the experiments. This research is part of the PhD
requirements for Natalia Belkin from Bar Ilan University
(BIU). Natalia Belkin (NB) was supported by a President’s
Fellowship from BIU and The National Fellowship Graduate
Program for Marine Conservation in the Mediterranean.
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Supporting information
Additional Supporting Information may be found in the online
version of this article at the publisher’s web-site:
Fig. S1. Weighted Venn diagram based on 16S rRNA gene
sequences comparing the initial bacterial composition (as
determined by OTUs) in control samples from T0 in mixed-
spring (red) experiment to samples from stratified-summer
(blue) experiment. The overlap of weighted Venn circles
reflects the OTU reads originating from the same lineage.
Circle sizes are proportional to the relative amount of each
sample’s OTUs in the data set. The accompanying table
details the percent of OTUs contributed by each season’s
data set, out of all compared OTUs per lineage (100%) and
the overlap of OTUs shared between the two seasons, as
was calculated for the comparative weighted Venn diagram.
The diagram was created using CoVennTree method
described by Lott et al. (2015).
Fig. S2. Weighted Venn diagram based on 18S rRNA
gene sequences comparing the initial eukaryotic phyto-
plankton composition (as determined by OTUs) in control
samples from T0 in mixed-spring (red) to samples from
stratified-summer (blue) experiments. The overlap of
weighted Venn circles reflects the OTU reads originating
from the same lineage. Circle sizes are proportional to the
relative amount of each sample’s OTUs in the data set. The
accompanying table details the percent of OTUs contributed
by each season’s data set, out of all compared OTUs per
lineage (100%) and the overlap of OTUs shared between
the two seasons, as was calculated for comparative
weighted Venn diagram. The diagram was created using
CoVennTree method described by Lott et al. (2015).
4120 N. Belkin et al.
Table S1. Changes in the abundance of ultra-phytoplankton
(Synechococcus, Prochlorochococcus and eukaryotic algae)
as obtained by flow cytometry. Detailed data are average cell
counts ± SD from triplicate mesocosms of each treatment at
the beginning (T0) and the end (after 11 and 12 days = Tend)
of the mixed-spring and stratified-summer experiments.
Table S2. Temporal changes in the average inorganic
nutrient concentrations ± SD measured from triplicate
© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 4105–4120
mesocosms of each treatment at the beginning (T0) and the
end (after 11 and 12 days = Tend) of the mixed-spring and
stratified-summer experiments.
Supporting Information Reference. Lott, S.C., Vob, B.,
Hess, W.R., and Steglich, C. (2015) CoVennTree: a new
method for the comparative analysis of large datasets. Front
Genet 6: 1–8.