Critical Review of Paper For Wastewater Class Project

CIVL 7230 PROJECT
Critical Review of Enhanced salinities, as

a proxy of seawater desalination
discharges, impact coastal microbial
communities of the Eastern
Mediterranean Sea
Article Authors: Natalia Belkin, Eyal Rahav, Hila Elifantz, Nurit Kress, and Ilana Berman-Frank
Review by: Brock Horsley

11/27/2015
Belkin, N., Rahav, E., Elifantz, H., Kress, N. and Berman-Frank, I. (2015), Enhanced salinities, as a proxy of
seawater desalination discharges, impact coastal microbial communities of the eastern Mediterranean
Sea. Environmental Microbiology, 17: 41054120. doi: 10.1111/1462-2920.12979
Objectives
Desalination plants discharge effluent brine into surface waters often at high
concentrations of salinity relative to ambient levels. While the environmental impact of
desalination effluent has previously been discussed, there are few laboratory studies on this
topic. Salinity has been considered the most important driver of the global distribution of
bacteria and other microbial life. The objective of the study was to perform a laboratory study to
examine the impact of increased salinity on planktonic microbial life in the coastal Eastern
Mediterranean Sea (EMS) of Israel.
Methods
Two experiments were performed: one from April 23rd to May 5th and another from July
7th to July 18th of 2013. The first one will be referred to as mixed-spring and the second as
summer-stratified. Time periods were selected to reflect different conditions in surface water
bodies. It was expected that initial conditions at each time period would influence microbial
environments. Both experiments contained three types of samples: a control seawater sample, a
5% added salinity sample, and a 15% added salinity sample. Three copies of each sample were
made for later statistical analysis. Each sample was a bag of seawater roughly 1 m3 in size, all in
a 16 m3 pool of circulated seawater. To account for the effects of isolation on microbial life in
each sample, the 5% and 15% added salinity samples were compared to the control samples. The
5% and 15% experiments were chosen to simulate conditions that were found at a nearby
desalination discharge site. The seawater was pumped from a site 300 m off of the coast and at a
depth of 2 m. To prevent evaporation, dilution, or contamination, the samples were covered with
polyethylene tops that still allowed gas exchange with the atmosphere. The tops also allowed for
50% surface light penetration.
1
Samples were taken from each bag two hours after brine was added. This time was
allotted so that each samples properties could be fully characterized. Each sample was
approximately 5 to 10 L, collected gravitationally for 11 to 12 days. Samples taken each day
were measured for salinity, temperature, chlorophyll (Chl a), bacterial productivity (BP), and
primary productivity (PP). DNA samples were also taken determine bacterial and eukaryotic
compositions. Samples were frozen until time to analysis to prevent decomposition. Quality
assurance was performed by national laboratories in the US, Canada, and Japan.
Results & Conclusions
Autotrophs experienced a decrease in photochemical efficiency, and heterotrophs saw
enhanced bacterial activity. Salt stress can inactivate the photosynthetic reaction centers and
inhibit protein synthesis. The data collected suggested that the immediate effect of salt addition
was salt stress on autotrophs and suppression of photosynthesis. For the 15% increased
experiment, PP decreased significantly in spring and summer tests. For mixed-spring, PP went
from 3.4 to 1.7 ug C/L/h, and stratified-summer went from 2.9 to 0.9 ug C/L/h. The decrease was
attributed to the immediate death of salt-sensitive phytoplankton. In heterotrophs, BP increased
by 2.5 times in mixed-spring and by 1.5 times in stratified-summer. The rapid BP increase was
attributed to the need of bacteria to self-regulate in response to the influx of salt. This includes
osmotic stress on cells, internal pH, and energetic potential of membranes. As for community
composition, increased salinity did not have a significant effect in mixed-spring when compared
to control. However, salinity was the dominant driver of community composition in stratifiedsummer. For the control in stratified-summer, bacterial communities decreased in diversity by
51%, while eukaryotic communities increased in diversity by 252%. In the 15% addition
experiment for stratified-summer, bacterial diversity decreased by 87% and eukaryotic diversity
2
decreased by 70%. In the 15% addition, cyanobacterial OTUs increased by 140%, while they
decreased by 90% in control. However, proteobacterial OTUs decreased by 30% in the 15%
addition, while they increased by 45% in control.
Although cyanobacterial OTUs increased in the 15% addition, Prochlorococcus marinus
(a major cyanobacterial order) disappeared after the first day of treatment. Studies have shown
that P. marinus contributes 9 to 18% of global marine carbon fixation. Among heterotrophs,
Pelagibacter declined significantly after six days in the 15% addition. Pelagibacter accounts for
roughly 30% of the bacteria in global oceans. It has been theorized that Pelagibacter consumes
osmolytes produced by P. marinus. Therefore, planktonic food webs near effluent sites of
desalination plants may change dramatically in the stratified-summer season of the EMS.
Turning to other microbial life, the diatom Gyrodinium was relatively low in abundance in
control during the stratified-summer experiment. In the 15% addition experiment, Gyrodinium
population was up 12% compared to other eukaryotic OTUs recorded. Some species of
Gyrodinium are known to form red tides and other harmful blooms. Thus, their increased
appearance in highly saline environments is of concern in desalination plant discharge sites.
The results of the experiments suggest that microbial communities have higher functional
plasticity in response to salinity in the mixed-spring, while they can be controlled by salinity
changes in the stratified-summer. Since biodiversity can help maintain the stability of an
environment to changes in the ecosystem, maintaining abundance of adaptive species can help
stabilize an environment that experiences change salinity. Salinity conditions can be a driving
factor in community composition of planktonic communities. An increase in salinity may reduce
species abundance, which would make to environment select for organisms that thrive in highsalinity environments. Seasonal changes in community composition can buffer against these
3
types of environmental stress. Ultimately, decrease in biodiversity that is caused by an increase

in salinity may destabilize the aquatic food web. The study recommended that other discharges
from desalination plants, such as coagulants and anti-scalants should be examined in future
studies.
Analysis
For strengths, the study had accurate modeling of the real-world scenario. The
concentrations tested were based on conditions at actual effluent sites. Seawater used was from
the EMS itself. The microbial community composition was seasonally consistent in spring and
summer. Continuous mixing was done to emulate sea mixing. The reporting had professional
statistical analysis and quality assurance. All tested means employed analyses of variance
(ANOVA). UNIFRAC Monte Carlo tests were used for comparing biological communities. The
study also took consideration for observational errors. All parameters were compared to controls.
Quality assurance was performed by national laboratories in the US, Canada, & Japan. They
assessed methodology for each parameter that had standardized testing. The study had good
explanations of tangible concerns, and provided direct links to the effects of increasing salinity.
Connections between change in microbial environment and known effects were made. The two
main connections were causation of red tides and changes in the food web. The study managed
to linked increases and decreases in microbial taxa to change in salinity. Species with elastic
response to change in salt will thrive, while some essential species in the local food web are
inelastic and become eradicated.
For shortcomings, the study presents other concerns about desalination plants with
limited discussion. It brings up how other substances in discharges from desalination plants may
be impact microbial communities. It seems to mention this as a major conclusion from the paper.
4
While it is a topic related to desalination plants, it is unrelated to the effects of changing salinity
in water. This concern should have presented as a side-thought, rather than among conclusions
from the research. Some of the laboratory methods did not have enough explanation for those
unfamiliar with the research. To those unaccustomed to lab techniques, some detailed were
understated or unclear. For instance, it was unclear how covers on experiment samples both
prevent contamination of atmospheric inputs but maintain gas exchange with the atmosphere.
Other details seemed unexplored, such as the effects of freezing the samples might have on
anticipated results.
Turning to limitations of the research, as desalination processes are a growing practice,
research into effects of discharge are not abundant. Many studies discuss implications of
increased salinity, but few actual lab studies exist. There are also few field measurements to
determine the impact of effluent discharge. It is hard to determine long-term conditions of
seawater when field data is limited. Since the study is contained to a 16 m3 zone, the effects of a
larger boundary zone are also unknown. It would be of interest to see if the same effects on a
microbial community would occur if there was no clearly defined boundary, as in a real seawater
scenario.
One of the unique findings of the paper involves how the increase in salinity led to
immediate decreased photochemical potential. A decrease in Chl a followed, reflecting death of
salinity-sensitive phytoplankton. It was previously known that salinity elevation leads to reduced
Chl a in some algae and cyanobacteria. However, there were no previous records found of rapid
changes within 2 hours of salt addition.
Critical Review of Enhanced

salinities, as a proxy of seawater
desalination discharges, impact
coastal microbial communities of
the Eastern Mediterranean Sea
Review by: Brock Horsley
Objectives
Objectives
Perform lab study on impact of increased
salinity on microbial community in Easter
Mediterranean Sea (EMS)
As a result of the discharge of high salinity from
desalination plants
Impact of desal plants has been discussed, few lab
studies
Methods
Methods
Two Experiments Performed
APR 23 MAY 5: Mixed-Spring
JUL 7 JUL 18: Stratified-Summer
Initial conditions influence environments
Methods
Each Experiment:
3 control samples
3 samples with 15% additional salinity
3 samples with 5% additional salinity
Chosen based on conditions at effluent sites found
in other studies
Methods
Approx. 1m3 in size for each sample type
16 m3 circulating pool
Pumped from a site 300 m off coast, depth 2 m
Polyethylene covers on each experiment
Prevent contamination but allow atmospheric exchange
Allowed 50% surface light penetration
Methods
Samples taken from each bag 2 hr after start
Taken in 5 to 10 L sizes, collected gravitationally
Collected for next 11 to 12 days
Parameters measured every day:
Salinity
Temperature
Cholorophyll (Chl a)
Bacterial productivity (BP)
Primary Productivity (PP)
DNA for determination of taxa

Autotrophs saw decrease in photochemical
efficiency
Heterotrophs saw enhanced bacterial activity
Expected, as salt stress can inhibit photosynthetic
reaction centers

15% addition experiment:
PP decreased significantly in spring and summer
3.5 to 1.7 ug/L in summer
2.9 to 0.9 ug/L in spring
Attributed to death of salt-sensitive
phytoplankton

15% addition experiment (for heterotrophs):
BP increased 2.5x compared to control in spring
BP increased 1.5x compared to control in summer
Attributed to need of bacteria to self-regulate due
to influx of salt
Osmotic stress on cells, internal pH, energy potential of
membranes

Community Composition:
Increased salinity had no significant effect in
spring compared to control
Salinity was dominant driver in summer
Summer Control:
51% decrease bacterial diversity
252% increase eukaryotic diversity
Summer 15% Addition:

87% decrease bacterial diversity
70% decrease eukaryotic diversity

Summer Experiment:
Cyanobacterial OTUs:
Control: decreased 90%
15% addition: increased 140%
Proteobacterial OTUs:
Control: increased 45%
15% addition: decreased 30%

Summer Experiment:
Although cyanobacterial
OTUs increased by 140% in
the 15% addition,
Procholorococcus marinus
disappeared after first day
of treatment
P. marinus is a major
cyanobacterial order
Contributes 9 to 18% of
global marine carbon
fixation

Summer Experiment:
Pelagibacter, a heterotroph,
declined 6 days later in the
15% addition experiment
Pelagibacter account for
roughly 30% of bacteria in
global oceans
Theorized that Pelagibacter
consumes osmolytes produced
by P. marinus
Conclusion: planktonic food
webs near effluent sites of
desalination plants may shift
profoundly in the stratifiedsummer season of the EMS

Summer Experiment:
Gyrodinium:
Control: relatively low (exact
number unreported)
15% addition: Gyrodinium
population up 12%
compared to other
eukaryotic OTUs
Some species of Gyrodinium
known to form red tides
Increased appearance of
Gyrodinium in high salinity
environments is of concern
at desalination discharge
sites

Final Conclusions:
Microbial communities have higher functional
plasticity to increased salinity in spring than in
summer
Maintaining an abundance of adaptive species can
help stabilize an environment that experiences
changes in salinity

Final Conclusions:
Decrease in biodiversity caused by increase in
salinity may destabilize aquatic food web
Recommendation for future study of other
discharges from desalination plants (coagulants,
anti-scalants) on microbial communities
Analysis
Strengths
Accurate modeling of real-world

scenario
Rather than random salt concentrations,
based on conditions at effluent sites
Pulled actual seawater from EMS for
experiment
Microbial community composition seasonally
consistent
Continuous mixing to emulate sea mixing
Professional Statistical Analysis and

Quality Assurance
Analyses of variance (ANOVA)
Provides a statistical test for multiple means to
determine statistical significance
To determine if a p-value is above significance
level
Probability of obtaining extreme results
UniFrac Monte Carlo tests for comparing
biological communities
Professional Statistical Analysis and

Quality Assurance
Consideration for observational errors
All parameters tested compared to controls
Quality assurance performed by national
laboratories in US, Canada, & Japan
Assessed methodology for each parameter
determination
Good Explanations of Tangible

Concerns and Link to Study
Connections between change in microbial
environment and known effects
Red tides
Change in food web
Linked increases/decreases in microbial taxa to

change in salinity
Species with elastic response to change in salt will
thrive
Some essential species in food web are inelastic /
eradicated
Weaknesses
Study Presents other Concerns with

Limited Discussion
Brings up how other substances in discharges
from desal plants may be of concern
Seems to mention is as a major conclusion from
the paper
It is a topic related to desal plants, but unrelated
to effects of changing salinity in water
Presentation of this concern should have been
presented as a side-thought, not among general
conclusions
Some Laboratory Methods UnderExplained

To those unaccustomed to lab techniques,
some detailed were understated
How do covers on experiment samples both
prevent contamination of atmospheric inputs but
maintain gas exchange with the atmosphere?
What effects did freezing the samples have on
anticipated sample results?
Limitations
Lack of Comparative Laboratory

Studies & Field Measurements
As desalination processes are a growing
practice, research into effects of discharge are
not abundant
Many studies discuss implications of increased
salinity, but few actual lab studies
Few field measurements of impact of effluent
discharge
Lack of Comparative Laboratory

Studies & Field Measurements
Hard to determine what to expect and longterm conditions of seawater when field data is
limited
Since study is contained to a 16 m3 zone,
effects of a larger boundary zone is unknown
Would we see the same effects on a microbial
community that is not allowed to migrate from a
more open boundary?
Unique Findings
Increase in salinity led to immediate

decreased photochemical potential
Decrease in Chl a followed, reflecting death of
salinity-sensitive phytoplankton
Previously known that salinity elevation leads to
reduced Chl a in some algae and cyanobacteria
No previous records found of rapid changes within
2 hours of salt addition
For 15% addition:
PP from 3.4 to 1.7 ug/L of Chl a in mixed-spring
PP from 2.9 to 0.9 ug/L of Chl a in stratified-summer
Knowledge Gaps
Lack of Labs Studies & Field Data

Increase in BP from salt addition not know to
be, but hypothesized to be, from elevated
organic consumption bacteria need to adjust
to osmotic stress on cells
Based on findings of a paper from (del Giogrio and
Bouiver, 2002)
Outside of first few hours, salinity elevations did
not significantly alter BP production
Effects of losing Pelgibacter and P.

marinus are speculative / leading
Noted that they are responsible for 9 to 18% of
marine carbon fixation
Does not note if other microbes that adjust to
highly saline environments that would outcompete these two would be able to replace them
Not clarified if influx of Gyrodinium is

really an issue
Argues that some species of Gyrodinium are
known to form red tides
Not specified which Gyrodinium species are found
in EMS
Unclear if the ones in this experiment would
actually contribute to red tide formation
Questions?
Article:
Belkin, N., Rahav, E., Elifantz, H., Kress, N. and Berman-Frank, I. (2015), Enhanced salinities, as a proxy of seawater
desalination discharges, impact coastal microbial communities of the eastern Mediterranean Sea. Environmental
Microbiology, 17: 41054120. doi: 10.1111/1462-2920.12979
Backdrops:
Image 1: http://teachmiddleeast.lib.uchicago.edu/foundations/geography/images/geography-05.jpg
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Environmental Microbiology (2015) 17(10), 41054120
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

Nurit Kress2 and Ilana Berman-Frank1*
1
Mina and Everard Goodman Faculty of Life Sciences,
Bar-Ilan University, Ramat Gan 52900, Israel.
2
Israel Oceanographic and Limnological Research,
National Institute of Oceanography, Haifa 31080, Israel.
Summary
Seawater desalination plants increase local coastal
salinities by discharging concentrated brine back to
the sea with 50% higher than ambient salinities. The
impacts of high salinities on microbial coastal populations of the eastern Mediterranean Sea (EMS) were
examined in two mesocosm experiments; first, during
the mixed-spring and second, during the stratifiedsummer periods with average salinity of 39. Ambient
salinities were increased by 5% and 15%. Higher
salinity (15%) mesocosms induced rapid (within 2 h)
declines in both primary productivity (PP) and algal
biomass parallel to an increase in bacterial productivity. Subsequently, for the duration of the experiments (1112 days), both Chlorophyll a and PP rates
increased (2 to 5 and 1.5 to 2.5fold, respectively)
relative to unamended controls. The initial assemblages of the ambient microbial populations and
intensity of salinity enrichments influenced the community responses. During the mixed-spring experiment, the composition of prokaryotic and eukaryotic
populations shifted only slightly, suggesting high
functional plasticity of the initial populations.
While during the stratified-summer experiment, high
salinity changed the composition and reduced the
biodiversity of the microbial communities. In an ultraoligotrophic environment such as the EMS, salinity
induced declines in microbial diversity may provide a
tipping point destabilizing the local aquatic food web.
Received 14 January, 2015; accepted 2 July, 2015. *For correspondence. E-mail ilana.berman-frank@biu.ac.il; Tel. (+972)-3-5318214;
Fax (+972)-4-6914842.
2015 Society for Applied Microbiology and John Wiley & Sons Ltd
Introduction
Large-scale seawater desalination is an effective solution to the freshwater shortage of many countries around
the world. Desalination in the Mediterranean Sea comprises 17% of the worlds total desalination and is one of
three semi-enclosed basins with intensive desalination
activity that are anticipated to raise ambient salinities of
the coastal habitats (Bashitialshaaer et al., 2011). In
Israel alone, seawater desalination by reverse osmosis
(SWRO) currently provides 450 million m3 (Mm3) y1
of fresh water along the easternmost Mediterranean
coastline. By 2025, water production supplied by five to
seven large-scale coastal plants is forecast to reach
750 Mm3 y1 constituting 30% of Israels freshwater
supply or c. 80% of the domestic and industrial needs
(Dreizin et al., 2008).
The main by-product of all desalination processes is the
large quantity of concentrated brine that is discharged
back to the marine coastal environment (Ahmad and
Baddour, 2014). With typical water recovery rates of
4050% in the desalination process, SWRO plants discharge brine to the sea with nearly twice the salt concentration of the ambient seawater. This discharge generally
includes other chemicals used in the process (e.g. coagulants, anti-foulants and anti-scalants) (NRC, 2008; UNEP,
2008; Spiritos and Lipchin, 2013). Along coastlines, water
loss due to its utilization by desalination plants, combined
with brine discharge back to the coastal environment,
increases the ambient salinity around the outfall areas
(Lattemann and Hpner, 2008). The temporal and spatial
dispersion pattern of the discharged brine differs among
sites and seasons due to the discharge technology of the
plant, changes in local currents and annual physical
chemical characteristics of the water column (Dawoud
and Al Mulla, 2012).
The long-term environmental and ecological impacts of
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
changes in the physical and/or chemical environment are
often followed by shifts in the composition and production
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
Temperature (C)
PO4 (mol L1)
NO2 + NO3 (mol L1)
Si(OH)4 (mol L1)
Diversity of bacterial species
Diversity of eukaryotic species
Chlorophyll a (g L1)
Primary productivity (g C L1 h1)
PSII quantum yield (Fv/Fm)
Bacterial productivity (g C L1 h1)
Bacterial abundance (105 cells ml1)
38.8 0.0
22.7 0.0
0.13 0.01
0.39 0.11
1.57 0.11
1762 513
528 138
0.24 0.02
3.36 0.42
0.12 0.01
0.89 0.61
6.3 0.7
40.5 0.1
22.7 0.0
0.12 0.02
0.55 0.21
1.69 0.11
1656 212
480 11
0.12 0.03
3.89 0.58
0.13 0.02
2.52 0.21
6.3 0.3
40.5 0.2
22.7 0.0
0.18 0.04
0.50 0.06
1.79 0.09
1729 179
651 31
0.17 0.01
1.77 0.24
0.07 0.03
2.26 0.30
5.7 0.3
39.3 0.2
29.9 0.2
0.02 0.00
BDL
1.78 0.08
939 322
173 74
0.24 0.02
2.86 0.39
0.13 0.00
2.34 0.37
4.2 1.1
41.3 0.4
29.9 0.1
0.02 0.00
0.21 0.12
1.86 0.01
1173 212
108 57
0.25 0.02
2.74 0.60
0.11 0.01
2.91 0.69
4.3 1.0
45.5 0.5
30.0 0.2
0.05 0.01
0.60 0.21
2.37 0.04
1299 122
131 19
0.17 0.01
0.86 0.15
0.06 0.02
3.26 0.80
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 gradients, as observed for the low-salt tolerance Mediterranean 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 effluents, laboratory studies or field measurements assessing
these impacts are scarce (Roberts et al., 2010; Elimelech
and Phillip, 2011; Liu et al., 2013). Moreover, scant information has been published showing the impacts of desalination 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 planktonic microbial populations from the coastal Eastern Mediterranean seawater (EMS) in 1 m3 mesocosms during two
1112 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 assemblages 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 (temperature 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 mol L1 PO4, 0.5 mol L1 NO2 + NO3, 0.59 mol L1
Si(OH)4) (Table 1).
Seasonality imprinted the initial nutrient concentrations
and microbial assemblages of autotrophs and
heterotrophs, and these differences affected the subsequent microbial community responses. At T0, both bacterial and eukaryotic representatives of the mixed-spring
experiment were more diverse than planktonic communities of the stratified-summer experiment (average effective 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 comprising each experiment with only 13.4% and 16.2%
2015 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 17, 41054120
High salinity impacts coastal microbial populations

respective identity in the composition of OTUs between
the mixed-spring community and stratified-summer community, with lower read numbers in the stratified-summer
experiment (Fig. S2).
The seasonal difference we detected in the OTU abundance and composition of in-situ phytoplankton communities 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 produces, along the Israeli coastline, a community comprised
of diatoms, dinoflagellates (micro-phytoplankton) and
nano-phytoplankton (320 m) (Azov, 1986). In the
stratified-summer experiment, the phytoplankton diversity
was lower (Table 1), reflecting the typical summer planktonic assemblages of the coastal Levantine waters dominated by the small (< 3 m) cyanobacteria and picoeukaryotic 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 community (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 g Chl a L1 and 3.15 g C L1 h1, respectively,
and Table 1) and may actually reflect the coastal origins of
the waters that are more uniformly mixed, receive terrestrial and anthropogenic nutrient inputs and depend to a
lesser extent than offshore populations on up-welled deep
nutrients.
In the EMS, heterotrophic bacteria contribute significantly to the food web structure and may compete successfully with phytoplankton when nutrients are limited
(Thingstad et al. 2005). Moreover, during thermal summer
stratification, a heterotrophic microbial food web supported by intensive recycling of organic carbon and nutrients 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 experiment than BP measured in the mixed-spring experiment
(2.3 g C L1 h1
vs.
0.9 g C L1 h1
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 addition, flow cytometry and molecular analyses revealed that
the microbial community structure remained unchanged
(Table 1, Table S1). Yet, rapid physiological responses
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 photosynthetic 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 photosynthetic 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 synthesis (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 g Chl a L1, 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 g C L1 h1 in the mixed-spring experiment and from
2.9 to 0.9 g C L1 h1 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. Moreover, 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 Mediterranean 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 consumption required by bacteria to maintain the changing
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 membranes (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 microbial 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 1.34C) 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 significantly lower than T0 only in the 15% treatments indicating 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 concentrations 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 important primary producers in the Levantine basin (Azov,
1986; Siokou-Frangou et al., 2010) with small (< 5 m,
pico-phytoplankton) cyanobacteria and larger (>5 m,
nano-phytoplankton) eukaryotic algae dominant along
the Israeli coast (Herut et al., 2012). By Tend, ultraphytoplankton biomass increased significantly in the 15%
treatments (P < 0.01; Fig. 1B), contributed mostly by
larger (>5 m) eukaryotic algal cell numbers (Table S1). In
the 15% higher salinity mesocosms these eukaryotic phytoplankton were probably diatoms as the stimulation of
algal biomass was accompanied by silica consumption.

At Tend silica concentrations (in the 15% treatment
mesocosms) were 5090% lower than T0 values and were
0.94 mol L1 and 0.11 mol L1 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 1.5fold 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 0.70.8) 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 phytoplankton grow under stress, e.g. nutrient limitation or
high light (Kolber et al., 1988), indicating that salinityinduced 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
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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 bacteria between seasons may have occurred due to the differing 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 communities and changes were primarily dictated by the
time of sampling.
In contrast, during the stratified-summer experiment,
salinity was a dominant driver impacting community composition 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 bacterial 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 9698% of all bacterial 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% additions caused a 30% decline).
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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
marinus), Oscillatoriophycideae and Nostocales. In the
15% treatment, P. marinus disappeared almost completely after 1 day, while the abundance of filamentous
cyanobacterial OTUs, specifically Oscillatoriophycideae
and Nostocales, known to adapt to high-salinity conditions
(Hagemann, 2011; Jeffries et al., 2012), increased compared with the controls (Fig. 3A, Fig. 4).
Among the heterotrophic bacterial groups, the
alphaproteobacterium (Pelagibacteraceae) Pelagibacter
and the gammaproteobacteria Altermondales declined
significantly after 6 days in the high-salinity (15%)
mesocosms of the stratified-summer experiment, (Fig. 3B,
Fig. 4). These were replaced by Rhodobacterales and
specifically Roseibacterium elongatum and Paracoccus
marinus of the Alphaproteobacteria (Fig. 3B), which are
aerobic,
chemoheterotrophic,
bacteriochlorophyllcontaining 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
Table 2. Temporal changes in microbial communities during the

stratified-summer experiment.
% Relative change Tend from T0
Salinity addition above control
16S
18S
Treatment
Control
5%
15%
True diversity
Distance (median)
Cyanobacteria OTUs
Proteobacteria OTUs
True diversity
Distance (median)
Diatom algae OTUs
Dinoflagellates OTUs
51%
0.32
90%
45%
252%
0.33
90%
300%
82%
0.42*
45%
13%
156%
0.39
86%
400%
87%
0.59*
140%
30%
70%
0.60*
15%
150%
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; calculated 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).
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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.
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 produced 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
during the stratified season of the eastern Mediterranean

(MayDec).
During the stratified-summer experiment, dominant
autotrophic eukaryotic groups (diatoms and dinoflagellates) 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 predominant 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
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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.

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 Mediterranean lagoon (Sarno et al., 1993).
Dinoflagellates comprised the second most abundant
group of eukaryotic phytoplankton in the summer experiment 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 dinoflagellate 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 microbial 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 functionally redundant microbial community that was controlled
by the salinity changes (Allison and Martiny, 2008). Biodiversity 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 facilitates processes such as recruitment, thus enhancing
recovery over longer timescales. Maintaining the abundance of species with an adaptive capacity, i.e. a combination 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).
4113
In contrast, a negative correlation between salinity and

diversity may diminish the community resilience to additional 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 stratifiedsummer experiment (Figs 4 and 5), may reduce the communities 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 communities. 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 longterm (chronic) input to the coastal areas probably maintains 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 predominately select for high-salinity resilient organisms.
Adaptation to salinity fluctuations is crucial for planktonic
organisms and acts as a decisive factor regulating functional 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) supported by cylindrical plastic frames were deployed within a
continuously circulating seawater 16 m3 pool to maintain
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N. Belkin et al.
ambient temperature and illumination. Surface coastal seawater was pumped to these mesocosm bags from Tel
Shikmona (Haifa, Israel) at 2 m depth, approximately 300 m
from the coastline (324934N, 345720E). 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 April5 May
2013 and (ii) 718 July 2013. These experiments were characterized 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 addition of artificial brine (80) prepared by dilution of seawater
salts (NeoMarine, 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 510 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 12 days at 08:00 am for a period of 1112
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
minimize biases due to sampling frequency causing a reduction 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-Daz et al.,
2011).
Laboratory analyses
Seawater for inorganic nutrient analyses (ortho-phosphate,
nitrate + nitrite, nitrite and silicic acid) was sampled into acidwashed 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 M, respectively,
while the limits of detection were 0.08 M, 0.008 M and
0.03 M respectively.
Chl a concentrations were determined by the nonacidification 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 m. The
pigments were extracted from the filters in 5 ml of 90%
acetone, at 4C, for 24 h in the dark. Chlorophyll a concentration 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 Ci of NaH14CO3 tracer
(Amersham, CFA3), and incubated for 4 h under ambient
irradiance and temperature (20.523.5C and 28.530C for
spring and summer experiments respectively). After incubation, particulate matter was collected on GF/F filter. The total
radioactivity in this fraction was determined by liquid scintillation (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 determined using a Fluorescence Induction and Relaxation
System (FIRe Satlantic, Halifax, Canada) to analyse the
photosynthetic characteristics of the autotrophic microorganisms (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 ms1 with 1 ms1 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 reaction 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 mmol1) at a final concentration of 100 nmol
Leucine L1. Samples were incubated in the dark at room
temperature (25 C), fixed and treated following the microcentrifugation protocol (Smith and Azam, 1992). Potential
bacterial production rates were calculated using a conversion
factor of 1.5 kg C mol1 with an isotope dilution factor of 2.0
(Simon and Azam, 1989).
Ultra-phytoplankton and bacterial abundances were enumerated by flow cytometry. Samples of 1.8 ml were immediately fixed after sampling with 5 l of 50% glutaraldehyde
(Sigma G-7651), incubated at room temperature for 10 min,
subsequently frozen in liquid nitrogen and kept at 80C until
analysis. Prior to the analysis, fixed samples were fast
thawed at 37C. Analysis was performed using a flow
cytometer FACScan Attune, fitted with argon lasers (405
and 488 nm). Beads of 1 m diameter (Polysciences) served
as standards (Marie et al., 2005; Stambler, 2006). The taxonomic 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 l 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 cell1 for Synechococcus
cells, 53 fg C cell1 for Prochlorochococcus cells and
2100 fg C cell1 for nanoeukaryotes (Campbell, 2001).
Statistical analyses
Prior to the statistical comparison, data were binned into two
groups: T0T5 and T6Tend, to reduce the effects of minor
observation errors. To assess treatment-dependent significant changes for each physiological parameter one-way
analyses of variance (ANOVAs) with permutations were performed 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 comparisons. For comparison of each parameter between different 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
4115
0.2 m pore size Supor filters (Pall Gelman, Ann Arbor, MI),
frozen in liquid nitrogen and kept at 80C until DNA extraction. Community genomic nucleic acids were isolated from
the filters, and media using a phenolchloroform extraction
method modified according to Massana and colleagues
(1997) and Brinkhoff and Muyzer (1997). For initial amplification, 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 reaction (PCR) was performed for next-generation sequencing
(Ion Torrent Life Technologies, USA) using specially designed
fusion primers with different tag sequences as: LinkerA-Tags27F 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 performed under the following conditions: 95C for 3 min followed by 25 cycles and 20 cycles (first and secondary PCR,
respectively) of 95C for 30 s; 60C for 30 s and 72C for
1 min and a final elongation step at 72C 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 analysed 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 coverage 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 checking was applied to remove chimera sequences, followed by
Lane mask to screen out hypervariable regions after alignment. 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 communities (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 community from different time points during the incubation period
(Moreno and Rodrguez, 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 examined 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 Presidents
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 publishers 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 mixedspring (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
samples OTUs in the data set. The accompanying table
details the percent of OTUs contributed by each seasons
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 phytoplankton 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 samples OTUs in the data set. The
accompanying table details the percent of OTUs contributed
by each seasons 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).

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
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: 18.

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CIVL 7230 PROJECT

Critical Review of Enhanced salinities, as

Review by: Brock Horsley

types of environmental stress. Ultimately, decrease in biodiversity that is caused by an increase

Critical Review of Enhanced

Results & Conclusions

Results & Conclusions

Results & Conclusions

Results & Conclusions

Results & Conclusions

Summer 15% Addition:

Results & Conclusions

Results & Conclusions

Results & Conclusions

Results & Conclusions

Results & Conclusions

Results & Conclusions

Accurate modeling of real-world

Professional Statistical Analysis and

Professional Statistical Analysis and

Good Explanations of Tangible

Linked increases/decreases in microbial taxa to

Study Presents other Concerns with

Some Laboratory Methods UnderExplained

Lack of Comparative Laboratory

Lack of Comparative Laboratory

Increase in salinity led to immediate

Lack of Labs Studies & Field Data

Effects of losing Pelgibacter and P.

Not clarified if influx of Gyrodinium is

Environmental Microbiology (2015) 17(10), 41054120

Enhanced salinities, as a proxy of seawater

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

4106 N. Belkin et al.

All parameters are averages SD of three mesocosms per treatment.

of biological communities. Sensitive coastal environments

mented two identical experiments: one in early spring

High salinity impacts coastal microbial populations

were recorded that were not a result of dilution. These

4108 N. Belkin et al.

osmotic stress of cells and to regulate the internal pH as

that was indicative of the adaptive capacity of the

High salinity impacts coastal microbial populations

from day 4 to Tend (P < 0.0005; Fig. 1D). No increase was

The major cyanobacterial order in all mesocosms was

Table 2. Temporal changes in microbial communities during the

High salinity impacts coastal microbial populations

Fig. 3. Temporal dynamics in the abundance

15% treatments is notable as Pelagibacter comprises

during the stratified season of the eastern Mediterranean

High salinity impacts coastal microbial populations

In contrast, a negative correlation between salinity and

Experimental design and sampling

High salinity impacts coastal microbial populations

DNA isolation and high-throughput

according to Jost (2007) using the conversion equation:

brine discharge from desalination. Int J Acad Res 3: 133

High salinity impacts coastal microbial populations

Kress, N., Galil, B., and Shoham-Frider, E. (2011) Monitoring

Marie, D., Partensky, F., Jacquet, S., and Vaulot, D. (1997)

High salinity impacts coastal microbial populations

4120 N. Belkin et al.

mesocosms of each treatment at the beginning (T0) and the