Biodegradation of Biodegradation of Emerging Contaminents Emerging Contaminents

by by Pseudomonas Pseudomonas butanovorabutanovora

Alisha NormanAlisha NormanMacArthur High SchoolMacArthur High School

Aldine Independent School DistrictAldine Independent School DistrictMentor: Kung-Hui (Bella) Chu/Civil EngineeringMentor: Kung-Hui (Bella) Chu/Civil Engineering

Summer 2008Summer 2008

The BackgroundIt 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 EstrogensEnvironmental 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 Endocrine-Disrupting CompoundsCompounds

• 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.

TricoslanTricoslan

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)

Prozac, antidepressant

17-estradiol

C

OH CH

HO

17-ethynyl estradiol

Diltiziam

OH

HO

Verapamil

What do you know aboutWhat do you know about

TriclosanTriclosan ??????? ???????

Health Health questionsquestions about BPAabout BPA

Aneuploidy: Down’sProstate cancer

Impaired brain development

Long-term memory formation

Weight control and obesity

Low sperm count

Dementia

Hyperactivity

200-300 genes200-300 genes affectedaffected

• 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.

Drinking Water Standards Drinking Water Standards

Engineered Water CycleEngineered Water Cycle

WaterUse

WaterTreatment

WastewaterTreatment

Water in

Nature

WaterTransport

Discharge/IndirectReuse

DirectReuse

WaterDistribution

WastewaterCollection

From Dr. Bachelor’s Notes

Pseudomonas butanovoraPseudomonas butanovoraAn aerobic G- bacteria isolated from activated sludge

(Sayavedra-Soto et al., 2001)

Believed to use these contaminants as a food source!

ResultsResults

ResultsResults

Isolation of BPA degraders

3.85ml of medium

0.15ml of 0.1g/l-BPA(15μg)

Allowed for equilibrium overnight at room at 150rpm

ATCC1581 + 5mM1-butanol+ 5mM

sodium citrate

30℃, 150rpm until OD600=0.8Wash twice

with ATCC1581

medium and centrifuge at

10,000rpm for 5min)

Resuspend in ATCC1581

medium with 5mM 1-butanol

(OD600=0.8)

30℃, 150rpm for 24

hrs

AutoclaveKilled cells

Resting cells

Resting cells with 5mM 1-butanol

Activated sludge from a

local wastewater

treatment plant (MLVSS:20,900

mg/l)

AS Only

n-butanol (5mM) enriched

Ethanol (0.5%) enriched

n-octane (0.2%) enriched

Dilution in NMS medium

MLVSS 500mg/l≒

Killed cells

Autoclave

Injection(1ml)

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)

0l .5mof 2g/l AS in NMS medium

+ 13μg/l of 8-2 FTOH → for 24 hrs

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.

After overnightat 30 , 150rpm℃

The Experiment

From the Lab to the Classroom!From the Lab to the Classroom!

The The BIGBIG Question??????? 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….The Big Question Part 2….• My plan is also to include the following TAKS

objectives related to Physics which are: 5A- demonstrate 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 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… The Next Question??? What is…

TurbidityTurbidity• 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 And how does it relate to Physics….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

SummarySummaryWe 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.

AcknowledgementsAcknowledgementsE3 Summer Research ProgramDr. Kung-Hui (Bella) ChuHyung Keun Roh, PHD CandidateMyung Hee Kim, 1st yr PHD studentNSF FundingTexas A&M UniversityAnd the Elite 8…..