Biodegradation of Emerging Contaminants by Pseudomonas butanorova

1. Biodegradation of Emerging Contaminants by Pseudomonas butanorova Shervada Hall Houston Independent School District Empowerment College Preparatory High School Mentor: Bella Kung Hui Chu/Civil Engineering

2. 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”

3. 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

4. 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.

5. Pharmaceuticals and Personal Care Products

6. 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 promotescross-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.

7. 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.

8. What can we do??!!!

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

10. Where can P. butanovera be found?

11. 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

12. 30 30 ℃ ℃ , 150rpm , 150rpm 30 30 ℃ ℃ , 150rpm until , 150rpm until Wash twice Wash twice for 24 hrs for 24 hrs OD OD =0.8 =0.8 with ATCC1581 with ATCC1581 Killed cells Killed cells Autoclave Autoclave 600 600 medium and medium and centrifuge at centrifuge at 0.15ml of 0.1g/l 0.15ml of 0.1g/l 0.15ml of 0.1g/l - - - P. Butanovera μ μ μ g) g) g) 10,000rpm for 5min) 10,000rpm for 5min) ATCC1581 ATCC1581 Resting cells Resting cells Resuspend Resuspend in in + 5mM1 + 5mM1 - - butanol butanol ATCC1581 medium ATCC1581 medium + 5mM sodium citrate + 5mM sodium citrate with 5mM 1 with 5mM 1 - - butanol butanol ( ( OD OD =0.8) =0.8) 600 600 Resting cells with Resting cells with 5mM 1 5mM 1 - - acetone l Injection Injection (1ml) (1ml) Dilution in NMS medium Dilution in NMS medium AS Only AS Only 3.85ml of medium 3.85ml of medium 3.85ml of medium After overnight After overnight Activated sludge Activated sludge at 30 at 30 ℃ ℃ , 150rpm , 150rpm from a local from a local Allowed for equilibrium Allowed for equilibrium Allowed for equilibrium wastewater wastewater n n - - butanol butanol (5mM) enriched (5mM) enriched overnight at room at 150rpm overnight at room at 150rpm overnight at room at 150rpm treatment plant treatment plant (MLVSS:20,900mg/l) (MLVSS:20,900mg/l) MLVSS MLVSS ≒ ≒ 500mg/l 500mg/l Ethanol (0.5%) enriched Ethanol (0.5%) enriched 0.5ml of 2g/l AS in NMS medium 0.5ml of 2g/l AS in NMS medium + 13 + 13 μ μ g/l g/l of 8 of 8 - - n n - - octane (0.2%) enriched octane (0.2%) enriched → → GC/FID analysis: GC/FID analysis: Autoclave Autoclave - - Agilent 6890N (FID) Agilent 6890N (FID) Several transfer by streaking on NMS agar slant Several transfer by streaking on NMS agar slant Killed cells Killed cells - - (30m (30m 250 250 m m 0.25 0.25 m HP 5MS column m HP 5MS column ⅹ ⅹ μ μ ⅹ ⅹ μ μ containing 8 containing 8 - - 2 FTOH (incubation at 30 2 FTOH (incubation at 30 ℃ ℃ ) ) Inlet temp=150 Inlet temp=150 ; detector temp= 300 ; detector temp= 300 ℃ ℃ ℃ ℃ - - 1 1 oven temp = 70 oven temp = 70 for 4 min, ramping at 30 for 4 min, ramping at 30 min min to 210 to 210 ℃ ℃ ℃ ℃ ℃ ℃ and and Growing visible colonies Growing visible colonies for 2 min for 2 min held at 210 held at 210 ℃ ℃ in NMS medium with 10mg/l of 8 in NMS medium with 10mg/l of 8 - - 2 FTOH (30 2 FTOH (30 ℃ ℃ ) ) - - Stock solutions of Stock solutions of P. butanova were prepared in acetone. - - Standards, from 1.5 Standards, from 1.5 - - 25 25 g, were prepared in medium g, were prepared in medium μ μ After continuous serial dilutions, After continuous serial dilutions, presumptive 8 presumptive 8 - - 2 FTOH 2 FTOH - - degraders degraders (r (r typically > 0.98) typically > 0.98) 2 2 were obtained and their 16S were obtained and their 16S rRNA rRNA genes were sequenced. genes were sequenced. Experimental Design 0.15ml of 0.1g/l-FTOHs (15μg) 0.15ml of 0.1g/l-FTOHs (15μg) 30℃, 150rpm for 24 hrs 30℃, 150rpm for 24 hrs 30℃, 150rpm until OD600=0.8 30℃, 150rpm until OD600=0.8 Wash twice with ATCC1581 medium and centrifuge at 10,000rpm for 5min) Wash twice with ATCC1581 medium and centrifuge at 10,000rpm for 5min) Killed cells Killed cells Autoclave Autoclave ATCC1581 + 5mM1-butanol + 5mM sodium citrate ATCC1581 + 5mM1-butanol + 5mM sodium citrate Resting cells Resting cells Resuspend in ATCC1581 medium with 5mM 1-butanol (OD600=0.8) Resuspend in ATCC1581 medium with 5mM 1-butanol (OD600=0.8) Resting cells with 5mM 1-butanol Resting cells with 5mM 1-butanol Injection (1ml) Injection (1ml) 3.85ml of medium 3.85ml of medium Dilution in NMS medium Dilution in NMS medium AS Only AS Only Allowed for equilibrium overnight at room at 150rpm Allowed for equilibrium overnight at room at 150rpm After overnight at 30℃, 150rpm After overnight at 30℃, 150rpm Activated sludge from a local wastewater treatment plant (MLVSS:20,900mg/l) Activated sludge from a local wastewater treatment plant (MLVSS:20,900mg/l) n-butanol (5mM) enriched n-butanol (5mM) enriched MLVSS≒500mg/l MLVSS≒500mg/l Ethanol (0.5%) enriched Ethanol (0.5%) enriched 0.5ml of 2g/l AS in NMS medium + 13μg/l of 8-2 FTOH → for 2 months 0.5ml of 2g/l AS in NMS medium + 13μg/l of 8-2 FTOH → for 2 months n-octane (0.2%) enriched 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) • 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 Autoclave Several transfer by streaking on NMS agar slant containing 8-2 FTOH (incubation at 30℃) Several transfer by streaking on NMS agar slant containing 8-2 FTOH (incubation at 30℃) Killed cells Killed cells Growing visible colonies in NMS medium with 10mg/l of 8-2 FTOH (30℃) Growing visible colonies in NMS medium with 10mg/l of 8-2 FTOH (30℃) After continuous serial dilutions, presumptive 8-2 FTOH-degraders were obtained and their 16S rRNA genes were sequenced. After continuous serial dilutions, presumptive 8-2 FTOH-degraders were obtained and their 16S rRNA genes were sequenced.

13. Summation World Find P. butanovera as an effective problem treatment source of estrogens Educational This information can be used in the classroom to bring awareness and interest in the environment

14. E3 Summer Research Program • Dr. Bella Chu • Myunghee Kim • Hyun Keun Roh