Biodegradation of Emerging Contaminants by Pseudomonas butanorova

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Biodegradation of Emerging Contaminants by
Pseudomonas butanorova

• Shervada Hall
• Houston Independent School District
• Empowerment College Preparatory High School
• Mentor Bella Kung Hui Chu/Civil Engineering

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• Ph.D University of California at
• Berkeley, 1998
• Assistant Professor Environmental Engineering
• 2 graduate courses and 1 undergraduate course
• Had work printed in over 13 publications such as
  Environmental Science and Technology, and
  Environmental Engineering Science

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Environmental Estrogens

• Found in sediments, rivers, lakes, drinking
  water, treated wastewater, and groundwater.
• Hormones and many other pharmaceuticals were
  detected in 108 (80 of 139) US rivers surveyed
  by USGS in 1999-2000. (Kolpin et al. 2002. ES T)
• Frequently detected compounds
• - Caffeine
• - Insect repellents
• - Hormones
• - Fire retardants
• - Plastizers

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Endocrine-Disrupting Compounds

• Endocrine system regulates important biological
  functions

• Growth Development
  Reproduction
• eating/sleeping fetus, puberty
  reproductive system

• Chemicals (synthetic or natural) mimic or act
  like hormones
• One of top six research priorities identified by
  EPAs Office of Research and Development in 1996.

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Pharmaceuticals and Personal Care Products

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Tricoslan

• A synthetic chlorinated aromatic compound with
  functional groups of ethers and phenols.
• Widely used as an antimicrobial agent in personal
  care, commercial, and medical products.
• Potentially promotes cross-resistance to
  antibiotics, toxic effects on ecological health,
  and formation of chlorodioxins from triclosan
  itself and its metabolites.
• Incomplete removal by wastewater treatment plants
  
• 79 biodegraded 15 absorbed to biosolids 6
  released into receiving water.

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• Plastic, epoxy resins, flame retardants, and
  other specialty products. Also used in the
  manufacture of products like eyeglass lenses,
  digital media, reusable food and drink containers
  and dental sealants.
• A weak endocrine disrupting compound detected in
  the environment, including wastewater.
• Toxic to aquatic organisms at concentrations of
  1-10 µg/L. BPA was found to stimulate the growth
  of rodent uterus.

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What can we do??!!!
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Pseudomonas butanovora

• An aerobic G- bacteria isolated from activated
  sludge
• (Sayavedra-Soto et al., 2001)
• Believed to use these contaminants as a food
  source!

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Where can P. butanovera be found?
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• World-Application
• Determine if P. butanorova
• can degrade Triclosan
• and/or Bisphenol-
• Improve our understanding
• of the fate of triclosan and
• Bisphenol A in wastewater
• and the environment.

• Educational- Application
• Help the teacher show
• how basic science
• concepts aid in solving
• real world problems.
• ex) dilutions scientific method

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Experimental Design
30?, 150rpm until OD6000.8
30?, 150rpm for 24 hrs
30?, 150rpm until OD6000.8
30?, 150rpm for 24 hrs
Wash twice with ATCC1581 medium and centrifuge
at 10,000rpm for 5min)
Wash twice with ATCC1581 medium and centrifuge
at 10,000rpm for 5min)
Autoclave
Killed cells
Autoclave
Killed cells
ATCC1581 5mM1-butanol 5mM sodium citrate
ATCC1581 5mM1-butanol 5mM sodium citrate
Resuspend in ATCC1581 medium with 5mM 1-butanol
(OD6000.8)
Resting cells
Resuspend in ATCC1581 medium with 5mM 1-butanol
(OD6000.8)
Resting cells
Resting cells with 5mM 1-butanol
Resting cells with 5mM 1-butanol
Injection (1ml)
Injection (1ml)
Dilution in NMS medium
Dilution in NMS medium
AS Only
AS Only
After overnight at 30?, 150rpm
After overnight at 30?, 150rpm
Activated sludge from a local wastewater
treatment plant (MLVSS20,900mg/l)
Activated sludge from a local wastewater
treatment plant (MLVSS20,900mg/l)
n-butanol (5mM) enriched
n-butanol (5mM) enriched
MLVSS?500mg/l
MLVSS?500mg/l
Ethanol (0.5) enriched
Ethanol (0.5) enriched
0.5ml of 2g/l AS in NMS medium 13µg/l of 8-2
FTOH ? for 2 months
0.5ml of 2g/l AS in NMS medium 13µg/l of 8-2
FTOH ? for 2 months
n-octane (0.2) enriched
n-octane (0.2) enriched

• GC/FID analysis
• Agilent 6890N (FID)
• (30m?250µm?0.25µm HP 5MS column
• Inlet temp150? detector temp 300?
• oven temp 70? for 4 min, ramping at 30?
  min-1 to 210 ? and held at 210 ? for 2 min
• - Stock solutions of FTOHs were prepared in
  ethanol.
• - Standards, from 1.5-25 µg, were prepared in
  medium
• (r2 typically gt 0.98)

• GC/FID analysis
• Agilent 6890N (FID)
• (30m?250µm?0.25µm HP 5MS column
• Inlet temp150? detector temp 300?
• oven temp 70? for 4 min, ramping at 30?
  min-1 to 210 ? and held at 210 ? for 2 min
• - Stock solutions of FTOHs were prepared in
  ethanol.
• - Standards, from 1.5-25 µg, were prepared in
  medium
• (r2 typically gt 0.98)

Autoclave
Autoclave
Several transfer by streaking on NMS agar slant
containing 8-2 FTOH (incubation at 30?)
Several transfer by streaking on NMS agar slant
containing 8-2 FTOH (incubation at 30?)
Killed cells
Killed cells
Growing visible colonies in NMS medium with
10mg/l of 8-2 FTOH (30?)
Growing visible colonies in NMS medium with
10mg/l of 8-2 FTOH (30?)
After continuous serial dilutions, presumptive
8-2 FTOH-degraders were obtained and their 16S
rRNA genes were sequenced.
After continuous serial dilutions, presumptive
8-2 FTOH-degraders were obtained and their 16S
rRNA genes were sequenced.
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Summation

• World
• Find P. butanovera as an
• effective problem treatment
• source of estrogens

• Educational
• This information can
• be used in the classroom
• to bring awareness and
• interest in the
• environment

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• E3 Summer Research Program
• Dr. Bella Chu
• Myunghee Kim
• Hyun Keun Roh