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

Ph.D University of California at Ph.D University of California at Berkeley, 1998 Berkeley, 1998 Assistant Professor Environmental Engineering Assistant Professor Environmental Engineering 2 graduate courses and 1 undergraduate course 2 graduate courses and 1 undergraduate course Had work printed in over 13 publications such as “Environmental Science and Technology”, and “Environmental Engineering Science” Had work printed in over 13 publications such as “Environmental Science and Technology”, and “Environmental Engineering Science”

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 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.

Pharmaceuticals and Personal Care Products Pharmaceuticals and Personal Care Products

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 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!

Where can P. butanovera be found?

World-Application Determine if P. butanorova can degrade Triclosan and/or Bisphenol- I 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

Experimental Design 3.85ml of medium 0.15ml of 0.1g/l-FTOHs (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 AutoclaveKilled cells Resting cells Resting cells with 5mM 1-butanol Activated sludge from a local wastewater treatment plant (MLVSS:20,900mg/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) 0.5ml of 2g/l AS in NMS medium + 13μg/l of 8-2 FTOH → for 2 months Several transfer by streaking on NMS agar slant containing 8-2 FTOH (incubation at 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 overnight at 30 ℃, 150rpm 3.85ml of medium 0.15ml of 0.1g/l-FTOHs (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 AutoclaveKilled cells Resting cells Resting cells with 5mM 1-butanol Activated sludge from a local wastewater treatment plant (MLVSS:20,900mg/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) 0.5ml of 2g/l AS in NMS medium + 13μg/l of 8-2 FTOH → for 2 months Several transfer by streaking on NMS agar slant containing 8-2 FTOH (incubation at 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 overnight at 30 ℃, 150rpm

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

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