Transcript Document

Phytoremediation of Antibiotics
Ninad Gujarathi
Department of Chemical Engineering
Colorado State University
PhD May 2005
Advisor: Professor James C. Linden
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
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
Plants Used in Phytoremediation Studies
• Aquatic species
- Pistia stratiotes (water lettuce)
- Myriophyllum aquaticum (parrot feather)
• Hairy root cultures
- Helianthus annuus (sunflower)
Water Lettuce
Parrot Feather
Sunflower Hairy Root Culture
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
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
General Structure of Tetracyclines
Two UV absorbing chromophores*:
1. A- chromophore ~ 270 nm
2. BCD-chromophore ~ 360 nm
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)
(UV abs.@355)/ (UV abs.@270)
A: 0.75;
B: 0.72
C: 0.64;
D:0.22
The modification appears to be
dominant at the BCD
chromophore
TC and OTC Degradation in Water or
Growth Medium
Fraction of initial
concentration
1.20
0.90
0.60
0.30
0.00
0.0
2.0
4.0
time, days
TC degradation
OTC degradation
Water Lettuce and OTC
Fraction of initial concentration
1.0
0.8
0.6
0.4
0.2
0.0
0.0
2.0
4.0
time, days
Live P. stratiotes exposed to 5 mg/L OTC
Root exudates from P. stratiotes exposed to 5 mg/L OTC
6.0
Fraction of initial concentration
Parrot Feather and OTC
1.0
0.8
0.6
0.4
0.2
0.0
0.0
5.0
10.0
15.0
time, days
Live M. aquaticum exposed to 5 mg/l OTC
Root exudates from M. aquaticum exposed to 5 mg/l OTC
Fraction of initial OTC
concentration
Sunflower Hairy Roots and OTC
1.00
0.75
0.50
0.25
0.00
0.0
2.0
4.0
6.0
time, days
Live roots exposed to 5 mg/L OTC
Root exudates exposed to 5 mg/L OTC
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
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
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
Oxidation of OTC
Potential mechanism
accounts for
- loss of electronic
resonance in
BCD rings
- loss of UV at 360 nm
- destruction of
antibiotic activity
Effect of Ascorbic Acid on OTC Modification
• Ascorbic acid (AA) is
common antioxidant
k, first-order rate constant
0.20
0.15
• Inhibition of OTC
modification increases
with increasing AA
concentrations
0.10
0.05
0.00
0
0.2
0.4
Concentration of ascorbic acid, mM
0.6
Fraction of initial OTC
concentration
Effect of ‘age’ of Root Exudates
• Rates of
modification
increased with age
of root exudates
1.00
0.75
0.50
0.25
0.00
0.0
0.5
1.0
1.5
time, days
3-weeks old
7-weeks old
5-weeks old
2.0
• ROS appear to be
the limiting
species in OTC
interaction
Effect of Salicylic Acid Elicitation in Water Lettuce
Microcosm on ROS Production
Control
Elicitation
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
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
Bioreactor System Layout
Hoagland’s
medium
OTC at 5
mg/L
Pond microcosm
Root
exudates
Treated water
Mixer
Bioreactor
Pond Microcosm
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
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.
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
% BOD removal in 2 days
40
38
36
32
30
25
20
10
0
0
L
LR
LRO LROS LROM
Acknowledgements
• Colorado State University Agricultural
Experiment Station (COL 00661)
• National Science Foundation (EEC0139478) Research Experience for
Undergraduates
– Byran J. Haney
– Heidi J. Park