Digital Moon

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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, Jaeweon Cho
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• Introduction
• Materials and Methods
• Results and Discussion
• Conclusions

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• 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
  (1993).
• Boron compounds in seawater do not dissociate
  into ions at low or neutral pH.
• Therefore, boron rejection in NF processes and
  SWRO desalination systems is low.
• At elevated pH, the rejection increases up to
  98-99 at pH 11 (Magara et al., 1998 Glueckstern
  and Priel, 2003).
• Boron rejection by RO membranes is affected by
  pH, permeate flux and temperature.

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• Introduction
• Animal and human studies of boron excretion
  suggested that boron homeostasis be maintained by
  urinary excretion (Sutherland et. al. 1999).
• 95 of the boron can be excreted from body within
  a week by urinary excretion.
• 19-fold variance in B intake (619 µg B/d to 11979
  µg B/d) coincided with a 37 difference in
  prostate cancer incidence (Barranco et. al.
  2007).
• 7-day treatments of Boric Acid (0-1000 µM) of
  DU-145 prostate cancer cells decreased Bcl-2
  protein production (Barranco et. al. 2007).

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• Materials and Methods
• Membrane Filtration-Solutions
• 5 mg B/L, model seawater and natural seawater
  solutions were used for membrane filtration
  processes.
• 5 mg B/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

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• Materials and Methods
• Membrane Filtration-Solutions
• Natural seawater samples were taken from coast of
  Masan City,Korea and prefiltered (0.45 mm).
  Cation concentrations of Masan seawater

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• Materials and Methods
• Membranes
• One Reverse Osmosis (RO), two Tight-Nano
  Filtration (NF) and one Loose NF membranes were
  selected in order to investigate boron rejection.
• Specifications of the membranes

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• Materials and Methods
• Membrane Filtration
• 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 (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).
• For pore size distribution experiments, 200 Da
  molecular weight Polyethylene Glycol (PEG)
  solution was filtered by membranes. Molecular
  Weight Cut Off (MWCO) and removal efficiencies
  were determined by HPLC-SEC apparatus.

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• Materials and Methods
• 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.
• All samples were analyzed by ICP-MS (Agilent,
  7500, US/An, octopole reaction system).

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• Materials and Methods
• Cell Culture
• Human embryonic kidney cells (HEK 293) were
  cultured in Dubeccos 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 ß-Actin protein
  production by using Bcl-2 and ß-Actin antibodies
  (Santa Cruz Biotechnology).
• Boron exposed and unexposed HEK 293 cells were
  measured on UV-Vis detector at 505 nm after 24
  and 48 h exposure to investigate boron effect on
  cell growth rate.

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• Materials and Methods
• Proteins Bcl-2
• 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.

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• Materials and Methods
• Proteins ß-Actin
• ß-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.
• Actin cytoskeleton effects the release of ROS
  from mitochondria and subsequent cell death
  (Gourlay and Ayscough, 2005).

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• Materials and Methods
• 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 ß-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).

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• Results and Discussion
• Membrane Characterization
• Zeta Potential

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• Results and Discussion
• Membrane Characterization
• Contact Angle

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• Results and Discussion
• Membrane Characterization
• MWCO

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• Results and Discussion
• Roughness of Membranes
• AFM

NE 70-Loose NF
NE 90-Tight NF
SR-RO
FL-Tight NF
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• Results and Discussion
• Roughness of Membranes
• SEM

NE 70-Loose NF
NE 90-Tight NF
SR-RO
FL-Tight NF
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• Results and Discussion
• Effect of Time on Boron Rejection

5 mg/L Boron solution
Model Seawater solution
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• Results and Discussion
• Effects of Solution and Operating Properties on
  Boron Rejection

Average Boron Rejection
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• Results and Discussion
• Effect of Boron on Cell Growth Rate

UV absorbance of HEK 293 cells after 24 and 48 h
of boron exposure
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• Results and Discussion
• Cell Culture
• Western Blotting

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• Conclusions
• Rejection of Boron by using membrane systems is
  stable for short term operations.
• Long term operations should be done in order to
  investigate fouling and other adverse effects of
  using natural seawater.
• Size Exclusion is one of the main mechanisms of
  boron rejection and is effected by flow regime,
  ionic strength.
• Other mechanisms which might effect rejection
  efficiency, such as diffusion and hydrophobic
  interaction should be investigated.
• Pressure is increased by using solutions which
  have high ionic strength. This leads more
  efficient rejections for FL (Tight-NF) and SR
  (RO) membranes.

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• Conclusions
• According to Western Blotting results Bcl-2 and
  ß-Actin protein production was not effected by
  boron concentrations 1 and 5 mg/L.
• More sensitive and accurate measurements for
  protein detection should be developed (LC/MS/MS).
• Different cell types and proteins should be
  investigated.

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• Questions ?
• Sarper Sarp sarper_at_gist.ac.kr
• Jaeweon Cho jwcho_at_gist.ac.kr