Biodegradation of Emerging Contaminents by Pseudomonas butanovora Alisha Norman MacArthur High School Aldine Independent School District Mentor: Kung-Hui.

Biodegradation of Emerging Contaminents by Biodegradation of Emerging Contaminents by Pseudomonas butanovoraPseudomonas butanovora

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

Environmental Engineering

•Protecting public health through application of engineering principles

Environmental engineers apply science and engineering principles to develop sustainable systems that protect human health and welfare and minimize the adverse effects of human activities on the environment. Environmental engineering involves:

•Providing safe, palatable and ample water supplies

•Disposing of or recycling wastewater, solid and hazardous wastes

•Predicting the transport and effects of contaminants in natural environments

•Managing hazardous wastes and remediating sites contaminated by improper disposal of such materials

Dr. Kung-Hui ChuDr. Kung-Hui Chu

Ph.D University of California at Berkeley, 1998Ph.D University of California at Berkeley, 1998

Assistant Professor Environmental EngineeringAssistant Professor Environmental Engineering

2 graduate courses and 1 undergraduate course2 graduate courses and 1 undergraduate course

Had work printed in over 13 publications such as Had work printed in over 13 publications such as “Environmental Science and Technology”, and “Environmental Science and Technology”, and “Environmental Engineering Science”“Environmental Engineering Science”

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 EstrogensEnvironmental Estrogens Found in sediments, rivers, lakes, drinking water, treated Found in sediments, rivers, lakes, drinking water, treated

wastewater, and groundwater.wastewater, and groundwater.

Hormones and many other pharmaceuticals were Hormones and many other pharmaceuticals were detected in 108 (80% of 139) US rivers surveyed by detected in 108 (80% of 139) US rivers surveyed by USGS in 1999-2000. USGS in 1999-2000. (Kolpin et al. 2002. ES &T)(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 functionsEndocrine 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.

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

The ResearchPlastics Exposure in Childhood• Plasticizers impart flexibility and durability…….Bisphenol A• Concerns exist about the potential todisrupt the endocrine system due tohormonal activity• Largely based on animal studies and littleresearch in human studies

Bisphenol A (BPA)

Summary of Findings

BPA is a weak estrogen• Animal studies:– Adverse birth outcomes– Male reproductive tract– Early pubertal development– Increased body size• Human studies– Limited human studies– Not classified with respect to carcinogenesis

Health Health questionsquestions about BPA about BPA

Aneuploidy: Down’s

Prostate cancer

Impaired brain development

Long-term memory formation

Weight control and obesity

Low sperm count

Dementia

Hyperactivity

200-300 genes200-300 genes affectedaffected

What can we do??!!!What can we do??!!!

Pseudomonas butanovoraPseudomonas butanovora

An aerobic G- bacteria isolated from activated sludge (Sayavedra-Soto et al., 2001)

Believed to use these contaminants as a food source!

Engineered Water CycleEngineered Water Cycle

WaterUse

WaterTreatment

WastewaterTreatment

Water in

Nature

WaterTransport

Discharge/IndirectReuse

DirectReuse

WaterDistribution

WastewaterCollection

From Dr. Bachelor’s Notes

Examine whether EDC’s can be Examine whether EDC’s can be degraded by butanol-oxidizing degraded by butanol-oxidizing bacteria.bacteria.

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

SummarySummaryWe desire our preliminary data to show that P. butanovora could degrade low concentration (0.5 mg/L) of bisphenol A.

The results of this study will improve our understanding of the fate of bisphenol A in wastewater and the environment.

That this information can be used in the classroom to bring awareness and interest in the environment

AcknowledgementsAcknowledgements

E3 Summer Research ProgramDr. Kung-Hui (Bella) ChuHyung Keun Roh, PHD CandidateMyung Hee Kim, 1st yr PHD student