Boron removal from seawater using NF and RO membranes, and effects of boron on HEK
293 human embryonic kidney cell with respect to toxicities
Sarper Sarp, Sungyun Lee, Xianghao Ren, Eunkyung Lee, Kyongmi Chon, Seok Ho Choi, Suhan
Kim, In S. Kim, Jaeweon Cho*
Department of Environmental Science and Engineering, Gwangju Institute of Science and
Technology (GIST), Oryong-dong, Buk-gu, Gwangju 50-712, Korea; *corresponding author: Tel.:
82-62-970-2443, Fax: 82-62-970-2434, e-mail: jwcho@gist.ac.kr
Abstract
Boron (B) is an important element which should be considered seriously for drinking water production.
Boron removal from seawater using Nanofiltration (NF) and Reverse Osmosis (RO) membranes was
investigated. Increase in salt concentration effects boron removal positively when RO membranes and
negatively when NF membranes were used. Time dependencies of membrane processes for boron removal
were also investigated. Based on the boron concentration in seawater and treated seawater, toxicity tests were
conducted using western blotting method. HEK 293, human embryonic kidney cell line and antibodies, Bcl-2
and β-Actin were used for boron toxicity tests.
Key Words: Seawater, Reverse osmosis, Nanofiltration, Boron Removal, Boron Toxicity, HEK 293 Human
Embryonic Kidney Cell, Bcl-2, β-Actin
1. Introduction
Seawater contains an average of 4.6 mg/L boron, but ranges from 0.5 to 9.6 mg/L ((Morgan, 1980; Woods,
1994). The World Health Organization has given a recommendation of boron concentration of less than 0.3
mg/L for drinking water guideline.
It is well known that boron compounds in seawater do not dissociate into ions at low or natural pH.
Therefore, boron rejection in NF processes and SWRO desalination systems is low and the process is not
adequate to produce permeate complying with the required quality standards (0.4-l.0 mg/l boron). At
elevated pH, the rejection increases up to 98-99% at pH 11 (Magara et al., 1998; Glueckstern and Priel,
2003). However, at high pH potential precipitation of calcium carbonate and magnesium hydroxide must be
avoided. Boron rejection by RO membranes is affected by pH, permeate flux and temperature. The boron
rejection of the currently applied SWRO systems, at nominal test conditions, is 85-90%. This corresponds to
about 78-80% boron rejection in operation of commercial SWRO systems (Busch et al., 2003).
Animal and human studies of boron excretion suggested that boron homeostasis be maintained by urinary
excretion (Sutherland et. al. 1999). Although approximately 95% of the boron can be excreted from body
within a week by urinary excretion, the remained part of the boron which is accumulated in body should be
investigated. Barranco et. al. (2007) reported that 19-fold variance in B intake (619 µg B/d to 11979 µg B/d,
depend on groundwater concentration) coincided with a 37% difference in prostate cancer incidence. They
also indicated that 7-day treatments of Boric Acid (0-1000 µM) of DU-145 prostate cancer cells decreased
Bcl-2 protein production. Bcl-2 is an integral inner mitochondrial membrane protein of relative molecular
mass 25,000 (25k) (Hockenbery et. al., 1990). Bcl-2 is one among many key regulators of apoptosis which
are essential for proper development, tissue homeostasis and protection against foreign pathogens (Lockshin
et al., 2000). Apoptosis plays a major role in normal organism development, tissue homeostasis, and removal
of damaged cells. Disruption of this process has been implicated in a variety of diseases such as cancer. The
localization of Bcl-2 to the inner mitochondrial membrane is novel for proteins with an oncogenic role. The
localization of Bcl-2 suggests that the main metabolic functions of the inner mitochondrial membrane, which
include oxidative phosphorylation, electron and metabolite transport, are involved in the survival mechanism
(Hockenbery et. al., 1990).
The objectives of this study are to investigate boron removal efficiency from model and natural seawater
using different types of NF and RO filtrations and effect of ionic strength on boron removal, and also to
investigate the boron toxicity to HEK 293 Human Embryonic Kidney Cell using western blotting method
with Bcl-2 and β-Actin proteins. β-Actin is one of six different actin isoforms which have been identified.
Actins are highly conserved proteins that are involved in cell motility, structure and integrity (Lambrechts et
al., 2004). Evidence from many organisms has shown that the accumulation of reactive oxygen species
(ROS) has a detrimental effect on cell well being. High levels of ROS have been linked to programmed cell
death pathways and ageing. Recent reports implicated changes to the dynamics of the actin cytoskeleton in
the release of ROS from mitochondria and subsequent cell death (Gourlay and Ayscough, 2005).
2. Materials and Methods
2.1 Membrane Filtration
5 mg B/L, model seawater and natural seawater solutions were used for membrane filtration processes. 5
mgB/L solution was prepared by dissolving boric acid (BA) in deionozed (DI) water. Model seawater was
prepared by dissolving sea salts (Sigma S9883) in DI water (Table 1). Natural seawater samples were taken
from cost of Masan City and prefiltered (0.45 mm). Cation concentrations of Masan seawater were given in
Table 2.
Table 1. Sea Salts (Aldrich).
TEST
SPECIFICATION
APPEARANCE
WHITE POWDER
SOLUBILITY
CLEAR COLORLESS SOLUTION AT 3.8GM PLUS 100ML OF WATER
CHLORIDE
19290 MG/L
SODIUM
10780 MG/L
SULFATE
2660 MG/L
MAGNESIUM
1320 MG/L
POTASSIUM
420 MG/L
CALCIUM
400 MG/L
CARBONATE/BICARBONATE
200 MG/L
STRONTIUM
8.8 MG/L
BORON
5.6 MG/L
BROMIDE
56 MG/L
IODIDE
0.24 MG/L
LITHIUM
0.3 MG/L
FLUORIDE
1.0 MG/L
NOTE
ALSO CONTAINS <0.5 MG/L OTHER TOTAL TRACE ELEMENTS
Table 2. Cation concentrations of Masan Seawater.
CATIONS
CONCENTRATION
Cu2+
365.12 µg/L
Ni2+
10.11 µg/L
Zn2+
226.95 µg/L
Cd2+
0.18 µg/L
Ag1+
0.75 µg/L
Pb2+
15 µg/L
Al3+
12.58 µg/L
Fe3+
12.77 µg/L
Mn2+
34.24 µg/L
As3+
2.90 µg/L
Sb3+
0.54 µg/L
Hg2+
0.22 µg/L
B
4.258 mg/L
Mg2+
1429.5 mg/L
Ca2+
40.27 mg/L
Membranes
Two Reverse Osmosis (RO) and two Nano Filtration (NF) membranes were selected in order to investigate
boron removal. Membranes were purchased from Saehan Industries Inc. Specifications of the membranes
were given at Table 3.
Table 3 Specifications of the selected membranes
Membrane
Manufacturer
NE70
Material
MWCO (Da)
Surface charge at pH 7
Classification
Polyamide
350
Negative
Loose NF
NE90
Saehan Corp,
Polyamide
200
Negative
Tight NF
FL
Korea
Polyamide
-
Close to Neutral
Brackish RO
Polyamide
-
Negative
Seawater RO
SR
Membrane Characterization
The membrane characterizations were done for four parameters including zeta potential, contact angle,
roughness and pore size distribution. The zeta potential values of the membranes were determined from
electrophoretic mobility measurements using ELS-8000 apparatus produced by Otsuka Electronics, Japan.
Contact Angle values of the membranes were determined by using Sessile Drop Method. Roughness of the
membranes were determined by using Atomic Force Microscope (AFM) and Scanning Electron Microscope
(SEM). In the pore size distribution experiments Polyethylene Glycol (PEG) solution was prepared from
PEG which has 200 Da molecular weight then this solution was filtered by membranes. Feed and permeate
samples were taken in order to investigate Molecular Weight Cut Off (MWCO) and removal efficiencies.
Samples were analyzed in HPLC-SEC apparatus.
Membrane Filtration
In membrane filtration tests, retentate flow rate and permeate flux were fixed to 500 ml/min and 8 µm/sec
respectively. Operating pressures were arranged in order to keep permeate flux constant. At 0, 30, 60, 90 and
120 minutes samples were collected from feed and permeate for 5 mg/L Boron solution and model seawater
solution. All samples were analyzed at ICP-MS apparatus.
Cell Culture
Human embryonic kidney cells (HEK 293) were cultured in Dubecco’s Modified Medium
(DMEM)(Invitrogen) supplemented with 10% FBS and Antibiotic-Antimycotic (Invitrogen). Every three
days cells were washed with PBS, trypsinized by TrypLE Express (Invitrogen) and re-seeded onto cell
culture plates (Nunc) with fresh media. 1 and 5 mg/L boron concentration were used to investigate boron
effect on HEK 293 cells. Boron was dissolved in media and cells were re-seeded with this media. After 4th
generation (4 re-seeding) with and without boron added media, Western Blotting was used to investigate
Bcl-2 and β-Actin protein production by using Bcl-2 and β-Actin antibodies (Santa Cruz Biotechnology).
Boron exposed and unexposed HEK 293 cells were measured on UV detector at 505 nm after 24 and 48 h
exposure to investigate boron effect on cell growth rate.
Western Blotting
HEK 293 cells were removed from culture plates by using TrypLE Express and soluble proteins were
extracted with RIPA buffer (Invitrogen) by centrifuging for 10 min at 8000 rpm. Extracted proteins were
mixed with sample buffer and reducing agent (Invitrogen) and exposed to NuPAGE 10% Bis-Tris Gel
(Invitrogen) by using XCell SureLock Mini-Cell (Invitrogen) for 20 min at 80V, followed by 1h at 200V.
Proteins were transferred to nitrocellulose membrane by iBlot (Invitrogen) transfer kit. Primary antibody
exposures (Bcl-2 and β-Actin) were applied for 16 hours, followed by 1 h secondary antibody exposure
(Goat Anti-Mouse IgG-HRP Conjugate (Zymax)). Membranes were subsequently submerged in ECL
detection reagent mixture (LG Health Care), wrapped in cassette (Fujitsu) and exposed to X-ray film
(Fujitsu).
3. Results and Discussion
3.1 Membrane Characterization
According to Zeta potential measurement, all membranes except FL membrane were negatively charged at
neutral pH (Figure 1). NF membranes (NE70 and NE90) are relatively hydrophilic than RO membranes
(Table 4) according to contact angle measurement. Sessile Drop Method was used to determine contact angle
values of membranes but for relatively hydrophilic membranes Captive Bubble Method might give more
accurate results. RO membranes showed better fractional removal than NF membranes in pore size
distribution measurements (Figure 2). Removal rate of 200Da PEG by membranes were given in Table 5.
NE90 membrane has highest MWCO according to removal rate of 200Da PEG. It was expected that SR
membrane would show best removal efficiency according to company acknowledgement but FL membrane
showed highest efficiency for removal of 200Da PEG. Roughnesses of membranes were determined by AFM
analyses (Figure 3 and Table 6) and it was found that NE70 membrane has the smoothest surface comparing
with the others. SEM pictures (Figure 4) were taken as further investigation of surface properties of
membranes. According to AFM and SEM pictures NE70 membrane has special surface structure.
pH
0
2
4
6
8
50
Zeta Potential (mV)
30
10
-10
NE 70
-30
NE 90
FL
-50
SR
-70
Figure 1. Zeta Potentials of selected membranes.
10
12
Membrane
Contact Angle
S.D
NE70
32.18
3.202
NE90
33.82
4.131
SR
57.54
1.600
FL
55.70
1.548
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
NE70
0
100
200
300
400
Molecular Weight (Da)
500
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Fractional Removal...
Fractional Removal...
Table 4. Contact Angle values of selected membranes.
600
0
50
100
150
200
250
Molecular Weight (Da)
300
350
1
Fractional Removal...
1
Fractional Removal...
NE90
0.8
0.6
0.4
0.2
FL
0.8
0.6
0.4
SR
0.2
0
0
0
100
200
300
Molecular Weight (Da)
400
0
500
100
200
300
Molecular Weight (Da)
400
500
Figure 2. Fractional Removal of 200Da PEG by selected membranes.
Table 5. Removal efficiency of 200Da PEG by selected membranes.
Membranes
NE70
NE90
FL
SR
Removal (%)
72.55
31.22
95.14
83.13
(a)
(b)
(c)
(d)
Figure 3. AFM pictures of selected membranes: (a) NE70, (b) NE90, (c) FL, (d) SR.
Table 6. Roughnesses of selected membranes according to AFM measurement.
Membranes
NE70
NE90
FL
SR
Roughness (nm)
17.86
67.51
78.16
61.41
(a)
(b)
(c)
(d)
Figure 4. SEM pictures of selected membranes: (a) NE70, (b) NE90, (c) FL, (d) SR.
3.2 Membrane Filtration
Boron removal efficiencies of membranes from Boron solution, model seawater solution and Masan
seawater were given in Figure 5 to 7. Although MWCO value of SR was lower than FL according to pore
size distribution, SR membrane showed most efficient boron removal. NF membranes showed lower removal
efficiencies than RO membranes. Operating pressure and MWCO values were propable reasons of lower
removal efficiencies by NF membranes. SR and FL membranes rewuired more operating pressures to
achieve 8 µm/sec permeate flux. Some of the membranes might have shoved overestimated boron removal at
0 min because of starting dilution. Comparison of average boron removals from different solution was given
in Figure 8. Boron removal efficiencies of NF membranes decreased by increasing ionic strength of the
solution and they showed better removal efficiencies with 5 mg/L boron solution. Boron removal efficiencies
of RO membranes were increased by increasing ionic strength. SR membrane showed better removal
efficiency with Masan Seawater and FL membrane showed better removal efficiency with model seawater.
Boron Removal Efficiency (%)
64
59
61
59
58
54
33
29
NE70
NE90
30
26
FL
SR
20
9
5
0
16
11
11
30
16
12
14
11
60
90
120
Time (min)
Boron Removal Efficiency (%)
Figure 5. Boron Removal efficiencies of selected membranes from 5 mg/L Boron solution.
74
74
69
8
37
9
1
0
NE70
NE90
42
38
36
30
27
75
74
30
33
FL
SR
10
5
60
4
3 3
90
120
Time (min)
Boron Removal Efficiency (%)
Figure 6. Boron Removal efficiencies of selected membranes from Model Seawater solution.
96
22
1
NE 70
6
NE 90
FL
SR
Figure 7. Boron Removal efficiencies of selected membranes from Masan Seawater.
Average Boron Removal Efficiency
96
100
90
Masan SW
80
73
Model SW
70
60
5 ppm B Solution
60
50
37
40
30
17
20
10
9 11
1
6
32
22
11
0
NE 70
NE 90
FL
SR
Figure 8. Average boron removal efficiencies of selected membranes from different solutions.
3.3 Cell Growth and Western Blotting
HEK 293 cell growths were investigated by UV absorbance at 505nm after 24 and 48 h exposure (Figure 9).
Boron addition improved cell growth according to UV absorbance for first 24 hours but became equal after
48 hours.
3.6
24 h
48 h
Absorbance at 505nm
...
3.2
2.8
2.4
2
1.6
1.2
0.8
0.4
0
Control
1ppm
5ppm
Figure 9. UV absorbance of HEK 293 cells after 24 and 48 h of boron exposure.
According to Western Blotting results Bcl-2 and β-Actin protein production was not effected by boron
concentrations 1 and 5 mg/L (Figure 10). More accurate result might be found by using LC/MS/MS analyses
for proteins.
Bcl-2
β-Actin
Acknowledgments
This research was supported by grant no. R01-2006-000-10993-0 from the Basic Research Program
of the Korea Science and Engineering Foundation (KOSEF), and also supported by the KOSEF
through the Advanced Environmental Monitoring Research Center (ADEMRC) at GIST.
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