1. Phytoremediation of Antibiotics Ninad Gujarathi Department of Chemical Engineering Colorado State University PhD May 2005 Advisor: Professor James C. Linden

2. Means for Countering Antibiotic Pollution of Wastewaters Convention methods: adsorption on hydrophobic beds, coagulation, and softening fail Reverse osmosis: successful but impractical for wastewater treatment Chemical and electrochemical oxidation: removes most antibiotics from wastewater Phytoremediation: an alternative for advanced oxidation methods

3. Phytoremediation Use of vegetation to contain, sequester, remove, modify or degrade pollutants from soil and water Pollutants: metals or organic compounds Metals either taken up or adsorbed on roots Organic compounds degraded or detoxified –before uptake by the plants –by agents released to the rhizosphere

4. Plants Used in Phytoremediation Studies Aquatic species - Pistia stratiotes (water lettuce) - Myriophyllum aquaticum (parrot feather) Hairy root cultures - Helianthus annuus (sunflower)

5. Water Lettuce

6. Parrot Feather

7. Sunflower Hairy Root Culture

8. Procedures for Obtaining Root Exudates Grow aquatic plants or root cultures in growth medium for the required amount of time Remove the plants/roots from the medium Filter the medium through a 0.2-micron filter Use immediately or refrigerate until convenient

9. Tetracycline and Oxytetracycline Antibiotics studied in this project Persistent in the environment Difficult to remove in wastewater treatment plants Can be oxidized by reactive oxygen species (ROS) –hydrogen peroxide –hydroxyl radical

10. General Structure of Tetracyclines Two UV absorbing chromophores*: 1.A- chromophore ~ 270 nm 2.BCD-chromophore ~ 360 nm

11. Evidence of OTC Modification by Root Exudates A- OTC in water (0 day) B- OTC in water (6 days) C- OTC in sunflower root exudates (0 days) D- OTC in sunflower root exudates (6 days) The modification appears to be dominant at the BCD chromophore (UV abs.@355)/ (UV abs.@270) A: 0.75; B: 0.72 C: 0.64; D:0.22

12. TC and OTC Degradation in Water or Growth Medium

13. Water Lettuce and OTC

14. Parrot Feather and OTC

15. Sunflower Hairy Roots and OTC

16. Summary All three plant systems gave significant antibiotic removal from water of both TC and OTC 1.Water lettuce  greatest removal rates (biomass concentration ~ 250 g/L) 2.Parrot feather  least removal rates, probably due to lower biomass concentrations (~20 g/L) 3.Sunflower hairy roots  intermediate removal rates, depending on period of growth

17. A Conclusion Filtered, cell-free and microbe free, root exudates gave comparable antibiotic removal rates Therefore: - physical adsorption to biomass was ruled out - uptake by the roots was ruled out - microbial interaction was ruled out - antibiotic interactions are with root secreted exudates

18. Discussion Aromatic compounds are known to be oxidized by plant root exudates Three possible sites of oxidation on the OTC molecule: OH groups in the A, B and D rings Quinone derivatives of OTC are the likely oxidation products

19. Oxidation of OTC Potential mechanism accounts for - loss of electronic resonance in BCD rings - loss of UV at 360 nm - destruction of antibiotic activity

20. Effect of Ascorbic Acid on OTC Modification Ascorbic acid (AA) is common antioxidant Inhibition of OTC modification increases with increasing AA concentrations

21. Effect of ‘age’ of Root Exudates Rates of modification increased with age of root exudates ROS appear to be the limiting species in OTC interaction

22. Effect of Salicylic Acid Elicitation in Water Lettuce Microcosm on ROS Production ControlElicitation

23. Discussion Plants respond to stress by producing ROS Sunflower root cultures and water lettuce produce greater ROS concentrations with elicitation using salicylic acid and methyl jasmonate Antimicrobial activity of the ‘modified antibiotics’ was determined to be destroyed

24. Design of Bioreactor System Water lettuce plants were employed in greenhouse microcosm Design constraints: Biomass concentration - 250 g/L Age of root exudates required - 7 days Reaction follows first-order kinetics OTC remediation in second stage bioreactor

25. Bioreactor System Layout Hoagland’s medium Root exudates Bioreactor OTC at 5 mg/L Treated water Mixer Pond microcosm

26. Pond Microcosm

27. Continuous Stirred Tank Reactor (CSTR) Shown as OTC remediation reactor –Temperature 30 C –Agitation 350 rpm –Residence time 7.5 h –Removal ~72% Also proposed as hairy root propagation reactor

28. Conclusions Antibiotics are oxidized by ROS produced by the plant roots The CSTR, when coupled with the pond microcosm, gave good OTC removal rates Phytoremediation of antibiotics is a possibility for treatment systems designed for field applications of the remediation system.

29. BOD Removal by Water Lettuce Root Exudates L = water LR = root exudates (RE) LRO = RE + OTC LROS = RE + 0.2 mM salicylate LROM = RE + 0.2mM methyl jasmonate

30. Acknowledgements Colorado State University Agricultural Experiment Station (COL 00661) National Science Foundation (EEC- 0139478) Research Experience for Undergraduates –Byran J. Haney –Heidi J. Park