Biodegradation of Emerging Contaminents by Pseudomonas butanovora

1. 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

2. Environmental Engineering 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 • Protecting public health through application of engineering principles

3. 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”

4. 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.

5. 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

6. 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.

8. 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

9. Bisphenol A (BPA)

10. 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

11. Health questions about BPA Impaired brain development Hyperactivity Aneuploidy: Down’s Prostate cancer Low sperm count Long-term memory formation 200-300 genes affected Dementia Weight control and obesity

12. What can we do??!!!

13. Pseudomonas butanovora An aerobic G- bacteria isolated from activated sludge (Sayavedra-Soto et al., 2001) Believed to use these contaminants as a food source!

14. Water in Nature Discharge/ Indirect Reuse Water Transport Direct Reuse Water Treatment Wastewater Treatment Wastewater Collection Water Distribution Water Use Engineered Water Cycle From Dr. Bachelor’s Notes

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

16. 30℃, 150rpm for 24 hrs 30℃, 150rpm until OD600=0.8 Wash twice with ATCC1581 medium and centrifuge at 10,000rpm for 5min) Killed cells Autoclave ATCC1581 + 5mM1-butanol + 5mM sodium citrate Resting cells Resuspend in ATCC1581 medium with 5mM 1-butanol (OD600=0.8) Resting cells with 5mM 1-butanol Injection (1ml) Dilution in NMS medium AS Only After overnight at 30℃, 150rpm Activated sludge from a local wastewater treatment plant (MLVSS:20,900mg/l) 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) Autoclave Several transfer by streaking on NMS agar slant containing BPA (incubation at 30℃) Killed cells 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. The Experiment 0.15ml of 0.1g/l-BPA(15μg) 3.85ml of medium Allowed for equilibrium overnight at room at 150rpm

17. 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

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