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Published in final edited form as:
Desalination. 2019 September 1; 465: 104–113. doi:10.1016/j.desal.2019.05.002.
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Bio-desalination of brackish and seawater using halophytic

algae
Endalkachew Sahle-Demessiea, Ashraf Aly Hassanb, Amro El Badawyc
aNational
Risk Management Research Laboratory, Office of Research and Development, U.S.
Environmental Protection Agency, Cincinnati, OH 45268, USA
bUnited Arab Emirates University, Department of Civil & Environmental Engineering, Al Ain,
United Arab Emirates
cCalifornia
Polytechnic State University, Civil and Environmental Engineering, San Luis Obispo,
CA 93407, USA

Abstract
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Global demand for water is rising. A sustainable and energy efficient approach is needed to
desalinate brackish sources for agricultural and municipal water use. Genetic variation among two
algae species, Scenedesmus species (S. sp.) and Chlorella vulgaris (C. vulgaris), in their tolerance
and uptake of salt (NaCl) was examined for potential bio-desalination of brackish water. Salt-
tolerant hyper-accumulators were evaluated in a batch photobioreactors over salinity concentration
ranging from 2 g/L to 20 g/L and different nutrient composition for their growth rate and salt-
uptake. During algae growth phase, the doubling time varied between 0.63 and 1.81 days for S. sp.
and 3.1 to 5.9 for C. vulgaris. The initial salt-uptake followed pseudo first order kinetics where the
rate constant ranged between !3.58 and !7.68 day!1 reaching up to 30% in a single cycle. The
halophyte algae S. sp. and C. vulgaris that were selected for pilot-scale studies here represent a
promising new method for desalination of brackish waters. Halophytic technologies combined
with the potential use of algae for biofuel, which offsets energy demand, can provide a sustainable
solution for clean, affordable water and energy.
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Graphical abstract
 Sahle-Demessie et al. Page 2
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Keywords
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Bio-desalination; Halophytic algae; Brackish water; Photobioreactor

1. Introduction
The rapid growth of population, the increase in per capita consumption, and the fast growth
of urbanization are creating critical water demand. The challenge of providing clean water is
on the rise [1,2]. New water-recovery technologies and improving the water communities’
infrastructure can partly alleviate the need. However, these approaches require long-term
investments and many developing countries are facing increasing chronic water problems
[3]. Thus, there is a need to improve available water resources at affordable costs to support
urban, rural and agricultural prosperity, and protecting the environment.

Desalination of brackish groundwater and seawater could unlock the vast water resource and
provide a sustainable source of water to water-stressed regions of the world. Thermal
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distillation and membrane separation account for 33% and 53% of the global utilization,
respectively [4]. However, the current desalination technologies consume large amounts of
energy (4–8 kWh of electrical power per m3 treated water) and require high capital
investment. The state-of-the-art reverse osmosis technology is reaching the thermodynamic
limit in the energy demand [4]. The cost of water produced by desalination is 3 to 5 times
the water produced from conventional sources [4], and the technology has yet to prove its
viability in providing water beyond household purposes and a few strategic industries at a
reasonable price. Thus, the supply of water from current desalination technologies to
agriculture may not achieve food security due to logistical, financial and environmental
problems [5]. This was still debated during the recent drought in California [6]. The
environmental impact of desalination technologies stems from 1) the high thermoelectric use
resulting in an emission of more than 1.8 kg CO2/m3 and 2) discharge of concentrated brine

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to the aquatic environment [7,8]. Therefore, there must be a paradigm shift that requires
other approaches.

Halophytic plants thrive in environments with high salt concentrations. Some halophile algae
are not only salt tolerant but can concentrate multiple times more salt levels than the water
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they live in [13]. Plants and algae that occupy estuaries and saline interior regions that have
high salinity habitats along salt and brackish marshes may undergo significant selections for
the capability to tolerate salt. This large pool of plant and algae provides many candidates
for use in engineered, biotechnology systems intended to treat saline water [9,10]. Although
halophyte plants are proven to remove salt from saline soils effectively [9], the data on their
use for treatment of saline water is scarce. The concept of bio-desalination is novel, and
research is still in its infancy. Macrophyte algae sequester salts in their frames and remove
them from solution [11]. The algae, cf. Pheridia tenuis, have shown to sequester salt within
the vacuoles of its tissues and convert the water to salt-free if it is left to grow in a bucket
that contained seawater [9]. The uptake of salts by halophyte represents the condition
wherein the salt is accumulated against an increasing osmotic gradient.

El-Sayed et al. [12] evaluated the growth of, Scenedesmus sp. (S. sp.), and reported grow
favorably and complete their life cycle even under full salinity level (35 g/L) and the algae
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removed at least 25% of the total salt. Duckweed in detention ponds tolerate salinity and
adapt with time to high salinity and have shown to reduce up to 25% of the salinity of the
water irrespective of the initial salinity [13]. In a previous study, S. obliquus was grown in
different salinities [14]. As the salinity of the water increased the desalination rate and the
removal of NaCl increased significantly. Salinity removal of up to 30% was reported after 16
days of incubation. Samples were centrifuged, filtered and then measured for total dissolved
solids. Another study reported similar cation uptake at salinities up to 25 using S. obliquus
[15]. The reduction of Na+ reached optimum levels (6.62 mg/L) at carbonate alkalinity of 35
mmol/L. Salinity stress intensified with increased concentrations of Na + for S. obliquus,
while it decreased significantly with higher concentrations of K+, Ca2+ and Mg2+.

Other algal species were shown to have the potential for biological desalination [16]. Thirty-
one shrimp ponds in areas of southern Thailand were sampled, and purple non-sulfur
bacteria (NW16 and KMS24) were isolated and investigated for bioremediation potential.
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Salinity removal of 30% was achieved together with other heavy metals [16]. A team of
researchers evaluated the use of cyanobacteria for the removal of sodium chloride from
seawater [17]. Two Euryhaline strains, one of freshwater (Synechocystis sp. Strain PCC
6803) and one of marine origin (Synechococcus sp. Strain PCC 7002) were identified as
possible candidates. Genetic modifications increased the bioaccumulation capacity of Cl! in
both strains. A thermodynamically favorable approach of growing cyanobacteria may be the
use of just sufficient light and nutrients for rapid growth while letting each cell enough
energy (in the form of sugars) to counteract the stress exerted by salt. Researchers studied a
dozen heterogeneous strains of euryhaline bacterial to determine whether salt-tolerant
bacteria have common characteristics that relate to both their taxonomy and the mechanism
of salt tolerance, concluded that these organisms are probably separate species of the same
genus [18].

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A pilot scale study was conducted in Egypt to evaluate the utilization of algae ponds in brine
desalination [19]. Algae (S. sp.) was grown under saline conditions and then introduced to a
saline basin at a rate of 0.4 L/basin/run, while growth media (BG-11) was supplied at a 0.1L/
basin/run. The volume of the basin was not mentioned in the article; however, it was
estimated at 420 l based on the analysis of the image presented in the article. The total
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dissolved solids concentration in the tank ranged from 40 g/L to 80 g/L. It was reported that
the system achieves high removal for salinity between 13% (for day one) to 63% (for day
six) and the removal efficiency increases with the increased salinity.

In this study, halophile algae (S. sp. and Chlorella vulgaris (C. vulgaris)) were grown in
photobioreactor (PBR) under carefully controlled conditions to sequester NaCl salt from
brackish and seawater, and their potential use for biological desalination was evaluated. The
biological mechanism and the factors affecting the reduction of salinity were investigated.
CO2 was utilized for the growth of algae, and valuable lipids were harvested from algae as a
byproduct that can be used to generate bio-fuel and supply energy back to the desalination
process. The growth rate of algae and the rate of salt uptake from seawater and brackish
water were measured. The impact of algae regrowth on salinity removal was also
investigated in a continuously stirred PBR with a filtration loop to maintain constant levels
of algae in the PBR. Finally, we explored the performance of a 60 L PBR scaled-up system
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that has with a filtration system in a recycle loop.

2. Materials and methods

2.1. Materials
The plan for the algal screening and bio-desalination study is summarized in Fig. S1
(Supplemental information).

2.1.1. Algae—Pure algae species samples were acquired from Culture Collection of
Algae at the University of Texas, Austin (UTEX). The cultures were grown in a culture
chamber at a temperature of 25 °C, a light intensity of 40 "mol m2 s!1 and 16/8 h day/night
cycle. Inoculation cell density of the cultures was reached by placing them in a stirred
incubator for two days at a constant light intensity of 280 "mol m2 s!1 and headspace
enriched with 0.5–5% carbon dioxide. All cultures were observed under an optical
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microscope for identification of species’ contamination. Despite the fact that C. vulgaris is
often found in freshwater, it is shown that it could resist salinity up to 20 g/L NaCl [20]
while no growth was observed at 30 g/L NaCl. Two commercial and a laboratory
synthesized growth media including F/2 (Kent Marine), Alga-Gro (Carolina Inc.), and
Biofilter-Media (BM) that was used earlier for growing aerobic bacteria in a biofilter
[21,22]. The cations concentration and percentage found in these growth media and artificial
seawater “Instant Ocean” are shown in Table S1. The nutrients added to the growth media
accounts for less than 3% of the total ions found in the saltwater. For every liter of brackish
water 20 mL, Alga-Gro was added, that contained 8 ppm of Na+ ion.

2.1.2. Preparing typical seawater—Artificial seawater was prepared by dissolving

mixed salts of known composition in deionized water. The ASTM Standard D1141-98
method (Table S2) has the advantage of being entirely chemically defined, reproducible and

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follows. However, preparing a solution, multiple components can be laborious and may not
support satisfactory algal growth. Commercial salt mix, Instant Ocean, was used to for an
artificial sea water that contained most of the major, minor, and trace elements necessary for
marine life. The salinity of Instant Ocean was compared with water containing NaCl.
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2.1.3. Nutrients—The algal culture media were prepared based on simulated seawater
supplemented with nutrients such as N, P, trace elements and vitamins, depending upon the
requirements of each species (Table S1). Physical factors such as pH and temperature were
carefully controlled during algal growth. Three growth media use for this study contain
nitrate concentration of 9, 47 and 283 ppm for Alga-Gro, F/2, and BM, respectively. The
BM has the necessary growth elements: B+3, Ca+2, Cl!, Co+2, Cu+2, Fe+3, K+, Mg+2, Mn+2,
Mo+6, NH4!, Na+, SO4!2, Zn+2, p-Aminobenzoic acid, Biotin, Thiamin Hydrochloride,
Cyanocobalamin, Folic Acid, Nicotinic Acid, Riboflavin, Pantothenic acid, pyridoxine
hydrochloride, and Thioctic Acid; in addition to a nutrient spike solution of 2 M NaNO3 and
0.22 M NaH2PO4#H2O. F/2 is similar into the composition of the BM, but it is missing B+3,
Ca+2, K+, and Mg+2. In addition, the vitamins available within the F/2 are very limited
namely, vitamin H, B1 and B12 (Tables S3 and S4). The type of growth media and salt
concentration modulate the pH of the solution (Table S5).
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2.1.4. Screening tests—Algae were grown in a temperature controlled PBRs that were
kept at 25 °C under 18/6 h fluorescent light cycle (Fig. S2). Air enriched with 0.5–5% CO2
was humidified in autoclaved deionized water and bubbled in tightly closed 250 mL and 500
mL bottles equipped with a vent. Different levels of salinity were achieved by adding either
synthetic seawater (Instant Ocean Co.) or sodium chloride (Fischer Scientific) to produce the
desired salinity. Algae were grown in salt water with salinity levels varying from brackish
water to salt water (2 to 20 mg/L of NaCl) and with three types of growth media nutrients.

2.1.5. Variation in carbon dioxide—The depletion of CO2 in dense cultures was

reduced by bubbling with air and increased CO2 concentrations (0.5–5%). Pure CO2 was
supplied through Praxair® compressed tanks fitted with a CONCOA® high purity regulator.
The amount of CO2 injected is controlled by a pH probe with an internal temperature
compensation. The PBR maintained at temperature for the microalgae growth. The air and
CO2 streams were first passed through inline 0.2 mm filter units (e.g., Millipore Millex GS)
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to maintain sterile conditions. Compressed air was used through a centrally housed system
to obtain a desired gas mixture ratio. The two sources of gas were connected to a Cole
Parmer® gas proportioner flow meter. According to the desired CO2 concentration, the gas
flow of house air and the compressed CO2 were adjusted and setting the mass flow
controller. However, unless there is a large amount of biomass taking up the CO2, the higher
concentrations could cause a significant decline in pH. The batch tests were run in
duplicates, and some of the optical density measurements were made at 1:5 dilutions for full
bloom samples.

A scaled up PBR was operated in closed container-based systems of cylindrical

photobioreactor (1.8 m long, 20.3 cm diameter, a volume of 60 l) with a recycle with the rate
of recycling of 27 L/min for successful algae growth and desalination. A circular light at the
top of the cylindrical reactor (Red and blue 90 W LEDs that has the spectrum of light, 630

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nm, 460 nm and an output 3800 lm) provided the best illumination for the recycle reactor
operation. For the pilot-scale system, multiple water quality monitoring sensors (YSI Yellow
Springs, OH, USA) were used for daily monitoring of the reactor including dissolved
oxygen, conductivity, temperature, turbidity, chlorophyll, pH, and an oxidation reduction
potential.
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2.2. Measuring algal growth

A set of 20 PBRs were continuously aerated and aliquots were withdrawn daily from the
PBRs and portions were centrifuged, and the supernatant liquid was analyzed for NaCl and
conductivity to determine the concentration between the cell and suspending liquid. Basic
cell culture management procedures, such as aseptic techniques and minimizing aerosol
contamination were implemented to avoid cross-contamination. Samples were collected in a
test tube from PBR and various measurements were made before they were returned to the
PBR bottles. Algal growth was measured using the change in turbidity (Hatch 2100),
chlorophyll (YSI 6025 chlorophyll sensor) and microscopic cell count of microalgae (Nikon
E600).

Conductivity was measured using two cell conductivity meters (Accumet AB150) without
dilutions, and the probe was recalibrated when the standard measurements deviated more
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than 5%. Nitrate ions and pH were measured using probes (Accumet). Sodium concentration
was also determined using ICP-OES; liquid samples were analyzed after filtration through
0.2 "m filter while solid samples were extracted from the filter and acid digested using nitric
acid. Chlorophyll concentration was determined by detecting the fluorescence in situ probe
with the YSI 6025 Chlorophyll sensor. Algal growth was monitored at an equal time interval
using a visible-light spectrometer at λ = 730 nm to track the density of the algal cells. These
species were tested under varying process conditions in finding their salt tolerance, growth
rate, and salt uptake.

2.3. Measuring waster salinity and salt uptake

The standard method for measuring primary and secondary anions according to U.S.
Environmental Protection Agency (EPA) Method 300.0 is the use of ion chromatography
(IC) [23]. Salinity can also be measured to be directly proportional to the amount of chlorine
in brackish and seawater, and because chlorine can be measured accurately by simple
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chemical analysis. Chloride probes salinity is measures based on chlorinity of the water.
Conductivity meter can also measure salinity very precisely and relatively easy to use
compared with the chemical techniques used to measure the chlorinity. Thus, electrical
conductivity is one of the necessary parameters of water quality detection in the seawater
desalination. Conductivity measures salinity of the water as a function of chlorinity relative
to standard seawater, thus the two approaches are similar [24]. In this study, electrical
conductivity and chlorinity were the primary approaches for tracking the extent of
desalination.

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3. Results
3.1. Initial assessment of algae growth
The initial batch experiments were aimed to explore the differences in algae growth and
salinity reductions based on the type and concentration of growth media used. The influence
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of the three types of growth media BM, F/2 and Alga-Gro for growth-nutrients was
compared at selected levels of initial salt concentrations using either Instant Ocean or pure
NaCl. The goal was to successfully acclimate the algae depending on ambient
concentrations and length of the exposure. During the acclimation stage, the algae
underwent a multistage process that included the readjustment of ionic and osmotic
potentials as well as broader physiological changes, including turgor adjustment in response
to salt stress [25]. Amezaga et al. [26], have proposed a possible mechanism for bio-
desalination using photosynthetic bacteria. The chemical energy carrier, ATP, powers Na+
export from cells either directly or indirect means by Na+-pumping ATPases or H+-ATPases
[27]. Environmental manipulations after the growth phase would halt Na+ export, and light
energy is used to absorb Cl! by halorhodopsin. The result of chloride in the cell is drawing
Na+ through permeable channel proteins [26].

The growth of algae was monitored based on the changes of turbidity over 40 days. Fig. 1
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shows the growth of S. sp. and C. vulgaris in different saltwater, dissolving either “Instant
Ocean” or NaCl, at selected concentrations of 2 g/L and 4 g/L, and two growth media. The
growth rate was influenced by the type of nutrients where the rate of growth followed the
order BM > F/2 > Alga-Gro (Fig. S3). Since Alga-Gro failed to show adequate algal growth
in these initial tests, it was removed from further analyses, and the results are not shown in
Fig. 1. This comparison is even more apparent based on chlorophyll concentration, where
the algae in BM turned dark green and can be distinguished by the naked eye (Fig. 2). The
effects of growth media were more significant at higher salt concentrations of 10 and 20 g/L
than at concentrations less than 4 g/L, where the BM showed superior growth compared to
F/2. S. sp. showed low growth rates at a salt concentration of 10 g/L using F/2, while growth
was not affected by an initial concentration of 20 g/L salt using the BM. Additionally, S. sp.
showed superior growth rates compared to C. vulgaris. This became more apparent at higher
salt concentrations. C. vulgaris growing at the salt concentration of 10 g/L showed low
growth rates. The influence of ions available in biomass is shown and growth media are
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shown in Figs. S3 and S4 (Supplemental Information).

The pH increased slightly with time regulating growth and causing some fluctuation and
lack of reproducibility. The growth nutrients added influenced the solution pH value with the
BM showing the highest pH at 8.7 for both algal species (Table S5). Both Alga-Gro and the
F/2 media showed an average pH below 8. On the other hand, the BM media has buffering
effect even with CO2 enriched air that lowers the water pH.

3.2. Kinetics of algal growth and salt uptake in a batch Photobioreactor

Algal growth is autocatalytic process such that the rate of growth is proportional to the cell
concentration already present. A general form of the growth equation is as follows:

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−1 𝑙𝑛𝑋2 − 𝑙𝑛𝑋1
𝜇 day = (1)
𝑡2 − 𝑡1

X1 and X2 are optical densities at time t1 and t2, and " is the instantaneous rate of increase
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[28]. This growth rate constant varied for the different types of algae, type of nutrients used,
and the initial salt concentration. For C. vulgaris the specific growth rate during the growth
phase varied from 0.07 to 0.21 per day that corresponded to doubling time of 3.1 to 5.9 days.
The growth rate of S. sp. was higher than that of C. vulgaris. For S. sp. the specific growth
rate during the growth phase varied between 0.63 and 1.82 per day that corresponds to
double time in the range of 0.42 and 1.2 days depending on the nutrient and salinity. The
highest specific growth rate was observed during the growing of S. sp. at a low salt
concentration of 2 g/L of Instant Ocean, while the lowest occurred for C. vulgaris growing in
a water solution of initial NaCl of 4 g/L.

3.3. Bioaccumulation and biosorption of NaCl in algal cells

There are many cellular functions such as nutrient uptake, photosynthesis, pH homeostasis
in an alkaline environment and nitrate assimilation that necessitate some Na+ [[29], [30],
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[31]]. However, high levels of Na+ concentrations can destabilize fatty acids in the cell
membrane and halt photo-autotrophic growth [32]. The level of sodium ion depends on the
strains of algae. The higher salt concentrations created unfavorable growth conditions in
algae. The increase in external concentrations of ions impairs the osmotic balance of the
cells and their surrounding and forces exosmosis from the cell. Exosmosis leads to the loss
of turgor pressure and tightens the influx of ions into the cells according to their
electrochemical gradients [25,26]. The algae acclimation proceeds to salt-tolerance and
gradually leads to steady-state growth.

The acclimation of algae to high salinity may include three basal processes: restoration of
turgor, regulation of the uptake and export of ions through the cell membranes, and
induction of the accumulation of photosynthetically produced osmoprotective “compatible”
solutes such as glycerol, and stress proteins. The influx of NaCl could disrupt the cellular
homeostasis and impair the function of biopolymers. The algae acclimation could involve
uptake and export of Na+ and K+ using redox-driven Na+ pump [27,29], for example,
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Dunaliella salina [30] accumulates protective compatible solutes and proteins [31,32], or
intercellular ion compartmentalization in the vacuole of the cell. If Na+ and Cl! are
sequestered in the vacuole of an algae cell, K+ and organic chemicals, such as proline and
glycine would accumulate in the cytoplasm of algae to balance the osmotic pressure [33].
Ahmad and Hellebust [34], suggested salt is accumulated within the cell for the species of C.
autotrophica to regulate the osmotic pressure.

Removal of salinity was observed based on conductivity measurements for S. sp. and C.
vulgaris species and all media types. As explained above, despite their salt tolerance and
sufficient growth, the considerable variation in salt uptake of the halophytic algae could be
as the result of their adaptation mechanism to salinity. As the concentrations of Na+ and Cl!
accumulated in the vacuole of the cells reach above 200 mM, and these concentrations could

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inhibit enzyme activities in the cytoplasm. However, obtaining direct experimental evidence
for the compartmentation of salts in halophytic algae is technically challenging even with X-
ray microanalysis.

Bioaccumulation and biosorption happen in most living organisms [35]. Metabolic

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absorption of salts in biological systems is a spontaneous process, where bioabsorption is

thermodynamically favored and requires no energy. The amount ion that a solid adsorbent
can remove from a solution is dependent on kinetics of the equilibrium and the composition
of the sorbents cellular surface [36]. The adsorption of ionic contaminants onto a cellular
structure could result in bioaccumulation that is a metabolic aerobic process that is driven by
energy from a living organism [36,37]. Microalgae can remove metal ions from water by
either biosorption or bioaccumulation [[38], [39], [40]]. The cell wall of algae is mainly
comprised of proteins, polysaccharides, and lipids. The cell wall is also negatively charged
and sticky that allows it to absorb cations [38]. The mechanisms that algae absorb cations
include surface deposition, physical and biosorption, active transport, and passive diffusion
[41]. However, the primary channel through which about 80% of cations are absorbed is
biosorption.

3.4. Endogenous salt uptake in algae

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Fig. 3 summarizes all the preliminary salt removal experiments with S. sp. and C. vulgaris
from the initial salt concentration 4 g/L. Results show the minimal difference in salinity
removal between both species. S. sp. showed average higher removal rates. Both species
underwent a phase of salinity decline initially until day 16. The following conductivity
measurement at day 18 shows an increase in salinity, followed by another phase of salinity
decline. The first drop occurred during the growth phase, while the second phase occurred
during the stagnant phase. The growth phase salt uptake had a steeper slope than the
stagnant phase salt uptake marking an initial high salt uptake. These results suggest that salt
is used in cellular uptake is mainly due to bioaccumulation and the salt uptake was not
merely a function of the number of cells. Salinity reduction increased when the generation of
new cells was much higher than cells lost. The slope in Fig. 3 during the stagnant phase
suggests that a fraction of the salt uptake is due to biosorption or the amount of salt released
from dead cells is less than the amount taken up in forming new cells. However, the increase
in salt concentration at day 18 marking the end of the growth phase is probably due to a
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sudden increase in the number of dead cells resulting salt release out of algal. This suggests
the process is biosorption.

The rate of salt uptake during the growth phase of the algae was estimated using first order
kinetics model:

Ln 𝐶 /𝐶𝑂 = 𝑘𝑡 (2)

where Co and C are concentrations of salt originally and at time, t.

The initial salt uptake rate for S. sp. varied between !0.015 to 0.0358 (day!1) depending on
the nutrient type and initial salt concentrations (Fig. S4). Two distinct regions could be
found in the relationship, the salt uptake part which corresponds to the algal growth phase

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and the salt release part which corresponds to the algal stationary phase. As the name of both
phases suggests, there is a high rate of salt uptake in the growth phase, while very mild
release occurs in the stationary phase.

Fig. 3 shows the effect of growth nutrients, salt type, and initial concentration on the
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reduction of salt concentration in the bulk phase. The growth of algae and removal of
salinity was modulated by the growth media and the initial levels of salt. Salinity reduction
up to 30% was observed using S. sp. grown in BM as seen in Fig. 4a. The salinity removal
for this alga in other test media was less than this value. The second highest removal amount
(26%) was observed with the C. vulgaris grown in BM as well. Comparable salt up-take was
observed for both algal species in the BM which indicated favorable algal growth conditions
are the main factor that determines the extent of salt removal. The presence of salt lowers the
activity of Ca2+. However, the BM allowed faster growth rate that resulted the higher
desalination rate could be attributed to the fact that increased concentration of Ca2+ in BM
solution countered the possibility of a NaCl-induced Ca2+deficiency in the cells. The
chlorophyll content of C. vulgaris increased in selected media in the following order: Alga-
Grow < F/2 < BM. The low chlorophyll indicates that the salt stress and nutrient deficiency
limited the algae to spend excessive energy for the synthesis of lots of new chlorophyll
molecules and binding proteins (Fig. S5). Fig. 4b. Highlights the importance of the
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contribution of other ions in salinity reduction, where the salinity of both algae species at
both initial concentrations of 2 and 4 g/L using Instant Ocean and NaCl salts are presented.
Instant Ocean showed significantly higher salinity removal rates with a more significant
margin at S. sp. compare to C. vulgaris. The results in Fig. 4c shows that the difference in
algae salt uptake between the 2 g/L and 4 g/L solutions were insignificant. Prescreening of
algal growth media for strain selection and the effects of different media on the performance
of algal-cultural under laboratory conditions is shown in Fig. 5.

3.5. Algae regrowth in a recycled media

The initial growth and salinity measurements suggested that dead cells release absorbed salt
back to the solution. Therefore, it is essential to avoid subjecting the solution to the decline
phase. Salinity removal was evaluated for both at S. sp. at 4 g/L for F/2 and BM, S. sp. at 20
g/L BM, and C. vulgaris at 4 g/L for both F/2 and BM. Algae underwent the growth,
stagnant phase, and started the decline phase. Results are shown in Fig. 5a. On day 35 after
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inoculation, all of the solution was centrifuged at 5000 rpm for 30 min. The concentrate was
removed, and it was then subjected again to the test conditions, where the growth nutrients
were added to the concentrate similarly to the starting conditions. The effects of growth
nutrients differ depending on the type of media, where F/2 media lead to a sharp increase in
growth reaching the peak at day 45 for S. sp. and day 55 for C. vulgaris. However, a short
growth peak was followed by immediate sharp decline. BM showed different pattern. While
the initial growth before day 35 was average the regrowth rate only led to an increase in
growth forward. The growth in some cases was double the initial growth indicating the algae
growth was successful in most of the test replicas. Regrowth was not observed in one of the
S. sp. with F/2 triplicates while it was successful in the other two bottles.

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Salinity removal is presented in Fig. 5b. Salt removal followed very similar pattern to the
initial results presented in Fig. 3. In the first 20 days, salinity removal up to 15% was
observed. Salinity was increased again around day 25 and it kept decreasing constantly
throughout the duration of the experiment. While the 4 g/L experiments showed relatively
close margins in performance, the 20 g/L didn’t show any salt removal until day 50. The
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maximum salt removal at 20 g/L salt was 3%.

3.6. Flow reactor with recirculation and system upscaling

Previous results have shown that the best salt removal was obtained during the growth phase.
Therefore, a method was devised to continuously filter out algae from the photobioreactor
system. A two liter PBR was seeded with S. sp. using BM for growth. The reactor was fitted
with a recirculation pump and the flow was forced through a course filter paper that operated
a rate of 2 L/min. The filter removed larger and aggregated cells and was replaced every
other day. This PBR was operated for a period of 3 months without the need to reseed. A
schematic of the setup and profile of the concentration decline is presented in Fig. 6. The salt
concentration was declining steadily till day 40 reaching 32% removal with an unexplained
peak that occurred for few days (14–17). After day 40 the salt concentration was almost
constant with a maximum removal observed of 36% at day 85. This system shows that a
continuous separation of algae works as well as regrowth in the same media. Both concepts
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showed superior performance suggesting that a mechanism of continuous algae removal

from solution is a key to success of this process. The performance of a three-stage
desalination process, based on the single growth cycle removal, is shown in Fig. S7.

Scaling-up the process to a 10-gallon (35 L) tank was unsuccessful for both types of algae S.
sp. and C. vulgaris. Algae was left to grow at a salt concentration of 10 g/L for 4 months.
However, blooming was not achieved in this larger volume. The salt concentration did not
register significant change during this period for both species. This was mainly due to
inadequate penetration of light within the aquarium.

3.7. Effect of algae on dissolved other ions

Salinity were measured based on the water conductivity and confirmed based on elemental
analysis using Inductively coupled plasma atomic emission spectroscopy (ICP-OES)
measurements. These measurements showed that the ratio of conductivity to Na+
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concentration was higher in Instant Ocean solution as compared to pure NaCl, which is due
to the contribution of other ions to conductivity. The conductivity measurement showed the
total ion concentration, but elemental analysis using ICP-OES proved algae grown in saline
water prepared from Instant Ocean had higher sodium removal rates as compared to solution
made from pure NaCl solution. This difference could be due the fact that Instant Ocean
contains other ions at the same total mass of salt in solution, which promoted algal growth.
The change in conductivity showed more salt removal by the S. sp. compared to C. vulgaris
(Fig. 3b), but the ion measurements showed that C. vulgaris is better in the removal of
sodium specifically than the S. sp.

In order to confirm the results of salt removal, algae ware separated from the solution by
centrifuge, and it was re-suspended in DI water. Ion measurements of these samples showed

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 Sahle-Demessie et al. Page 12

Na+ to be the most abundant ion in the biomass. By extrapolating the results obtained from 5
mL liquid sample to the total liquid volume of 250 mL, removal of ca. 10% was observed
within all samples. Comparing these results with the quantities of salts sequester because of
the small amount of the biomass available in such small liquid volume. However, the result
provided an additional proof of the salt removal and its uptake within the alga cells.
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The distribution of ions found in both species is shown in Fig. S3. Sodium was the most
abundant ion in both species followed by potassium. Barium, calcium, iron, phosphorus,
sulfur and zinc are available in relatively high amounts. S. sp. showed higher tendency to
retain sodium and potassium, which are the most abundant ions, however similar amounts
were observed for the rest of the ions. The amount of sodium retained by the S. sp. was the
same from solution based on Instant Ocean and pure NaCl, while all the other ions were
higher in Instant Ocean. The same behavior was observed in liquids containing C. vulgaris.
On the other hand, using F/2 for growing S. sp. yielded higher sodium, potassium and
phosphorus retention than the BM. Whereas for C. vulgaris using BM showed highest
sodium and other major ions followed by F/2 and finally Alga-Gro.

3.8. Strategies of algae-water separation

Once bio-desalination process has occurred, efficient algae-water separation is needed. The
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downstream separation process of algae cells from the desalinated water without affecting
the integrity of the cells and unintended release of absorbed salt back into the desalinated
water could be a technical and an economical challenging bottleneck in the bio-desalination
process [[42], [43], [44], [45]]. The challenges of separating algae cell from aqueous
suspensions come from due to little density difference to water, the microscale size with
negatively charged surfaces that stabilizes cell suspensions and excess extracellular
polymeric substances. To destabilize colloidal suspensions of algae coagulation, filtration
and dissolved air floatation were tested. Coagulants such as polyelectrolytes or polyhydroxyl
complexes have shown to have superior properties of attaching segments of algae to vacant
sites of other algal particles forming a 3-D floc network.

4. Conclusion and assessment integrated bio-desalination and biofuel

process
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The provision of adequate water supply to match the word’s growing demands has become a
major challenge. The difficulties of the traditional source of water will continue to grow as
we become more dependent on groundwater from steadily falling aquifers. In this paper we
have presented exploratory study on the challenges and opportunities associated with the use
of algae in reducing salinity of brackish water. The current physicochemical desalination
technologies have negative impacts on climate, ocean salinity and other natural processes.
The use of algae to remove salinity while providing necessary algal growth have much lower
energy demand while the algae can be harvested for either to generate biofuel or source for
protein. Biodesalination requires an efficient and a low-cost separation of algal cells from
water (Table 1). Successful use of bio-desalination technology could ensure a sustainable
supply of water for drinking and agricultural use at low in cost while producing valuable
bio-fuel as a byproduct to supply energy for the desalination process.

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A flowchart for algal screening for bio-desalination is presented in Fig. S7. The total energy
for desalination of water can be reduced by making use of harvested algae for energy
generation and integrating the process with conventional membrane desalination. The
integration of bio-desalination with biofuel production could address the water-energy and
has the potential to be a sustainable approach to reinventing drinking water (Fig. S7). The
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success of developing a biological desalination depends on the selection of algal species that
can grow in seawater, the optimization of salt-uptake, the separation of algae from the
treated water in a sustainable manner.

Life cycle assessment of the integrated desalination system of algae bio-desalination

preceded by reverse osmosis (RO) is necessary for a holistic assessment of the system.
Experimental studies are essential to examine the effect of the bio-desalination on the
organic matter concentration that could increase the potential for biofouling reverse osmosis
membrane.

To design a realistic industrial facility, we extrapolated laboratory measurements and

combined them with known processes developed for bio-fuel and desalination system. The
potential salinity reductions were computed based on various scenarios and were used to
select the process chain. In addition to the overall energetic balance of the production chain
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the impacts of the reduction of water salinity, further work is needed to the benefits of algal
bio-fuel compared to diesel fuel, and issues of water quality related to large-scale algal
growth.

This study revealed that the successive optimization of the bio-desalination process and the
potential use of algae for desalination and biofuel resource could make this technology
attractive and sustainable. The application of multiscale cascaded PBRs and continuous
harvesting of the algae to sustain the high algae growth rate would increase the salt uptake.
This technology could be used either for desalination of brackish water with lower salinity
or as a pretreatment for seawater prior to reverse osmosis. Bio-desalination could be used for
multiple purposes depending on the initial salt concentration. In a descending order with
respect to salt concentration, the target applications of this technology are influent for
conventional desalination, animal feed, irrigation, and drinking purposes, respectively. With
further development such as multistage operation, this technology could be an
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environmentally sustainable method that serves multiple purposes to provide water that is
safe, appropriate for agricultural use, and produce biofuel. As is the case in other
biotechnological processes, bio-desalination requires efficient separation of algae from the
water. Floatation and filtration are suitable methods to remove up to 99% of algae. However,
the presence of algal biomass requires an integrated bio-desalination process which is likely
to build on knowledge of both algal bioreactors and innovative water treatment processes.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

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 Sahle-Demessie et al. Page 14

Acknowledgement
The research received seed funding through the EPA internal competition “Pathfinder Innovation Projects”
challenge in pursuit of high-risk, high-reward research ideas. We thank Tom Deinlein for building the laboratory
PBRs. AAH was a post-doctoral research fellow at the National Risk Management Research Laboratory
administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the
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U.S. Department of Energy and the U.S. Environmental Protection Agency.

Disclaimer

The US EPA, Office of Research and Development funded and managed this research described herein. It has been
subjected to the Agency’s Administrative review and has been approved for external publication. Any opinion
expressed in this paper are those of the authors and do not reflect the views of the U.S. EPA. Approval does not
signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.

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Highlights
• Halophytic technologies provide a sustainable solution for clean and
affordable water.
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• Scenedesmus species and Chlorella vulgaris are examined for potential bio-
desalination of brackish water.

• The initial salt-uptake follows pseudo first order kinetics.

• Pilot-scale introduces a novel promising method for desalination of brackish

waters.
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Fig. 1.
Growth of algae measured based on change in algal turbidity in brackish water with Instant-
Ocean and NaCl initial 2 g/L and 4 g/L (a) S. sp. using BM, (b) S. sp. using F/2 Media, and
(c) C.C. vulgaris using F/2 growth media, (d) C.C. vulgaris BM.

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Fig. 2.
Biodesalination experimental system using S.S. sp. (a) a column PBR, (b) PBR of Alga-Gro
media, (c) PBR of BM (d) PBR of F/2 growth media, (e) Microscopic images of Halophytes
Species Tested (UTEX Culture collection of Algae).n
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Fig. 3.
Reduction of normalized conductivity for S. sp. and C. vulgaris grown at selected conditions
where the C/Co is shown using BM growth media, at initial salt concentration, 4 g/L.

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Fig. 4.
Salt removal showing the effects of growth media of F/2 and BM on (a) S. sp. (b) C.
vulgaris, influence of salinity types using BM growth media on (c) S. sp. and C. vulgaris,
and the effects of initial concentration (d) S. sp. and C. vulgaris.

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Fig. 5.
Desalination using selected algae S. sp. and C. vulgaris regrowth tests based on changes in
(a) turbidity, (b) salinity. Legend symbols stand I - S. sp., F/2 media, 4 g/L, II - S. sp., F/2
media, 4 g/L, III - S. sp., F/2 media, 20 g/L, IV - C. vulgaris, F/2 media, 4 g/L, V - C.
vulgaris, BM, 4 g/L.

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Fig. 6.
Biodesalination performance of brackish water using S. sp. grown in a recycle pilot-scale
recycled PBR.

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Table 1.

Summary of salinity reduction at end of desalination and algal removal.

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Sample Algal cell concentration Chloride concentration Decreased in Cl! %

Cell/mL mV

Full bloom ~104 12.4 21

Centrifuge ~102 7.2 58

Centrifuged and filtered Negligible 3.2 74

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