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 (Kolpin et al 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.

What do you know about Triclosan ???????

Health questions about BPA Aneuploidy: Down’s Prostate cancer Impaired brain development Long-term memory formation Weight control and obesity Low sperm count Dementia Hyperactivity genes affected affected

EPA has established National Primary Drinking Water Regulations or Primary Standards. –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

Engineered Water Cycle Water Use Water Treatment Wastewater Treatment Water in Nature Water Transport Discharge/ Indirect Reuse Direct Reuse Water Distribution 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

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

Isolation of BPA degraders 3.85ml of medium 0.15ml of 0.1g/l- BPA(15μg) Allowed for equilibrium overnight at room at 150rpm ATCC mM1- butanol + 5mM sodium citrate 30 ℃, 150rpm until OD 600 =0.8 Wash twice with ATCC1581 medium and centrifuge at 10,000rpm for 5min) Resuspend in ATCC1581 medium with 5mM 1- butanol (OD 600 =0.8) 30 ℃, 150rpm for 24 hrs Autoclave Killed 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) (30m ⅹ 250μm ⅹ 0.25μm 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 μg, were prepared in medium (r 2 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 overnight at 30 ℃, 150rpm The Experiment

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: 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 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 ]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 /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, 1 st yr PHD student NSF Funding Texas A&M University And the Elite 8…..