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

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 Chu 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”

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. 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) 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 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.

The Research Plastics Exposure in Childhood Plasticizers impart flexibility and durability …….Bisphenol A Concerns exist about the potential to disrupt the endocrine system due to hormonal activity Largely based on animal studies and little research 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 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

What can we do??!!!

Pseudomonas 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 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

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

Summary We 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

Acknowledgements E3 Summer Research Program Dr. Kung-Hui (Bella) Chu Hyung Keun Roh, PHD Candidate Myung Hee Kim, 1 st yr PHD student