Transcript Biodegradation of Pharmaceuticals and Personal Care Products by

Biodegradation of
Emerging Contaminents
by Pseudomonas
butanovora
Alisha Norman
MacArthur High School
Aldine Independent School District
Mentor: Kung-Hui (Bella) Chu/Civil Engineering
Summer 2008
The Background
It was recently reported that 80% of
surveyed streams (about 108 US
streams were contaminated with
trace amounts of pharmaceuticals
and personal care products including:
•steroidal hormones
•antimicrobial agents
•Stimulants
•and many other organic componds.
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
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
EPA’s Office of
Research and
Development in 1996.
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.
 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.
PPCPs
• More than 80 different classes
(Daughton and Ternes, 1999. Environ. Health Perspect. 107:907-938)
Diltiziam
OH
C
CH
HO
17-ethynyl estradiol
Prozac, antidepressant
OH
HO
17-estradiol
Verapamil
What do you know about
Triclosan ???????
Health questions about
BPA
Impaired brain development
Hyperactivity
Aneuploidy: Down’s
Prostate cancer
Low sperm count
Long-term memory formation
200-300 genes
Dementia
affected
Weight control and obesity
Drinking Water Standards
• EPA has established National Primary
Drinking Water Regulations or Primary
Standards.
http://www.epa.gov/safewater/mcl.html
– legally enforceable standards that apply to
public water systems.
– Primary standards protect public health by
limiting the levels of contaminants in drinking
water.
Engineered Water Cycle
Water
Transport
Water
Treatment
Water
Distribution
Water
in
Nature
Direct
Reuse
Water
Use
Discharge/
Indirect
Reuse
Wastewater
Treatment
Wastewater
Collection
From Dr. Bachelor’s
Notes
Pseudomonas butanovora
An aerobic G- bacteria isolated from
activated sludge
(Sayavedra-Soto et al., 2001)
Believed to use these contaminants as
a food source!
Results
Results
The Experiment
Killed cells
0.15ml of
0.1g/lBPA(15μg)
Autoclave
Resting cells
Injection
(1ml)
Resting cells with
5mM 1-butanol
30℃,
150rpm
for 24
hrs
Wash twice
with
ATCC1581
medium and
centrifuge at
Resuspend in 10,000rpm for
5min)
ATCC1581
medium with
5mM 1butanol
(OD600=0.8)
30℃, 150rpm until
OD600=0.8
ATCC1581
+ 5mM1butanol
+ 5mM
sodium citrate
Dilution in NMS medium
AS Only
3.85ml
of
medium
Allowed for equilibrium
overnight at room at
150rpm
After overnight
at 30℃, 150rpm
n-butanol (5mM) enriched
MLVSS≒500mg/l
Ethanol (0.5%)
enriched
Isolation of BPA degraders
0l .5mof 2g/l AS in NMS
medium
+ 13μg/l of 8-2 FTOH
→ for 24 hrs
n-octane (0.2%)
enriched
GC/FID analysis:
Agilent 6890N (FID)
(30mⅹ250μmⅹ0.25μm HP 5MS column
Inlet temp=150℃; 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 > 0.98)
Killed cells
Activated
sludge from a
local
wastewater
treatment plant
(MLVSS:20,900
mg/l)
Autoclave
Several transfer by streaking on
NMS agar slant containing BPA
(incubation at 30℃)
Growing visible colonies
in NMS medium with 10mg/l BPA
After continuous serial dilutions, presumptive
BPA-Degraders were obtained and their 16S
rRNA genes were sequenced.
From the Lab to the Classroom!
The BIG Question???????
• How are Physics and Environmental
Engineering Related?
• TEKS….
• The core elements I would like to transfer back to the classroom are
the importance of planning and implementing investigative
procedures including asking
• questions, formulating testable hypotheses, and selecting
equipment, and technology. Additionally collecting data and make
measurements with precision, organizing, analyzing, evaluating,
making inferences, and predicting trends from data, and finally
communicating valid conclusions.
• These elements are inclusive but not limited to
TAKS objectives: 2A,2B,2C, and2D.
The Big Question Part 2….
• My plan is also to include the following TAKS
objectives related to Physics which are: 5Ademonstrate wave types and their
characteristics through a variety of
• activities such as modeling with ropes and coils,
activating tuning
• forks, and interpreting data on seismic waves.
• 5B demonstrate wave interactions including
interference, polarization,
• reflection, refraction, and resonance within
various materials.
The Big Question Part 3…
• The following TEKS I would
like to include are as
follows:
• 2A 2B 2C 2D 2E
• 8A 8B 8C
The Next Question??? What is…
Turbidity
• What is Turbidity?
Turbidity is a measure of the amount of
suspended material in the water. It describes the
clarity of water. Suspended materials in water,
such as clay, silt, and algae, reduce water clarity
and cause turbidity.
And how does it relate to
Physics….
• Technically, turbidity is an optical
property of the water based on the
amount of light reflected by
suspended particles
• Basically, this means that turbidity
is closely related to the amount of
light scattered at 90 degrees when
a light source is shined through a
sample.
•
•
Turbidity Test
LESSSON SHEET [ Student Handout ]
•
Time Frame: one period
•
Concept:
Turbidity is the amount of muddiness or cloudiness in the water usually caused by stirring up of
sediments. Turbidity affects the amount of sunlight reaching aquatic plants and therefore affects the
entire aquatic food chain by affecting the amount of oxygen available. Oxygen content of aquatic
ecosystems is perhaps the single most important factor in the ecological health. Lower oxygen reduces
biodiversity. Many factors including turbidity, affects oxygen content. These include eutrophication
caused by unnaturally high nutrient levels, temperature, plant growth, surface area.
Objective:
Students will conduct a test to determine the turbidity of water samples.
•
Materials:
•
•
LaMotte Turbidity test kit (price: $5.50/call: Frey 1-888-222-1332/kit#: F22366)
Preparation:
Collect a water sample from a local body of water. This turbidity test will not work unless the water is
fairly turbid. Accurate laboratory turbidometers are too expensive for most secondary school science
budgets. Therefore if the water sample is not turbid enough, "create" a sample by adding a little
cornstarch to the water. If you do this do not telecommunicate your turbidity data. The lab will only serve
to introduce the concept of turbidity.
Procedure:
•
Fill one of the tubes with the water sample to the 50 ml mark.
•
Fill the second tube with tap water to the 50 ml mark.
•
Looking down into the tubes from the top, compare the black dots on the bottom of the tube (if one
cannot be seen, only fill each tube to the 25 ml mark).
•
Using the eye dropper, add the standard turbidity reagent .5ml at a time, until the dots appear the same.
Record the amount of reagent used.
•
Each milliliter of reagent equals 10 Jackson Turbidity Units (JTU's) if the 50 ml line was used. Each
milliliter equals 20 JTU's if the 25 ml line was used. Use the enclosed chart if you need more help
calculating the JTU's.
Turbidity
Summary
We desired our preliminary data to show that P.
butanovora could degrade low concentration (0.5
mg/L) of bisphenol A and Triclosan.
The results of this study will improve our
understanding of the fate of bisphenol A
and Triclosan in wastewater and the
environment.
That this information can be used in the
classroom to bring awareness and interest in the
environment
That students will appreciate understand study of engineering
concepts.
Acknowledgements
E3 Summer Research Program
Dr. Kung-Hui (Bella) Chu
Hyung Keun Roh, PHD Candidate
Myung Hee Kim, 1st yr PHD student
NSF Funding
Texas A&M University
And the Elite 8…..