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Clint Richardson and Enric Bonmati PowerPoint Presentation
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Clint Richardson and Enric Bonmati

Clint Richardson and Enric Bonmati

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Clint Richardson and Enric Bonmati

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  1. Phytoremediation of TNT Contaminated Soils using Nitrate Reductase Enzyme Extract from Spinacia oleracea Clint Richardson and Enric Bonmati Department of Civil and Environmental Engineering New Mexico Tech

  2. Introduction • Research Focus and Objectives • Materials and Methods • Proposed Kinetic Model • Results and Discussion • Conclusions

  3. Introduction • Worldwide Use of Explosive Energetics • Munitions, Construction, and Mining, etc. • Major Problem: Military Training Installations • Formerly Used Defense Sites (FUDS) • Nitro-Energetic Compounds (NECs) • TNT (2,4,6 trinitro-toluene) • RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) • HMX (octahydro-1,3,5,7tetranitro-1,3,4,7 tetrazocine)

  4. Introduction • Environmental Concern • Soil and Groundwater Contamination • Mass. Military Reservation on Cape Cod, Mass. • Ft. Wingate near Gallup, New Mexico • EPA Health Advisories • Drinking water criteria for TNT and RDX based on a lifetime exposure cancer risk level of 1 in 106 • No general soil criteria exists to date

  5. Introduction • TNT Molecular Structure (C5H7N3O6) Ref. McFarlan (2000)

  6. Introduction • TNT Sequential Reduction • TNT  ADNT  DANT  TAT • ADNT • 2 amino-4,6 dinitrotoluene • 4 amino-2,6 dinitrotoluene • DANT • 2,4 diamino-6 nitrotoluene • 2,6 diamino-4 nitrotoluene • TAT • 2,4,6-triaminotoluene Ref. Medina and McCutcheon (1996)

  7. Introduction • TNT Oxidation/Reduction Transformation Ref. McFarlan (2002)

  8. Introduction • Phytoremediation • Use of live plants or components of plants to degrade or transform a contaminant • Plants use a variety of enzymatic reactions inside the cell structure to grow and reproduce • e.g. nitrate reductase enzyme for nitrate metabolism • Spinaciaoleracea • High content of nitrate reductase enzyme • Capable of interacting with NACs • Commercially available or easily grown

  9. Research Focus and Objectives • Focus • Remediation of TNT contaminated soil • Use of an enzyme extract from Spinacia oleracea • Objectives • To develop an in-situ cost-effective, environmentally safe alternative to traditional ex-situ methods of soil remediation, such as composting or incineration • To define a workable protocol for field application of the enzymatic treatment method

  10. Research Focus and Objectives • Experimental Approach • Evaluate overall TNT transformation using • Aqueous phase microcosms • Soil-slurry phase microcosms • Unsaturated soil microcosms

  11. Research Focus and Objectives • Experimental Approach (cont’d) • Characterization of Spinacia oleracea extract • Nitrate reductase activity • Protein content • Evaluation of TNT transformation kinetics • Identify appropriate kinetic model • Integrate kinetics with nitrate reductase activity • Determine efficiency of transformation

  12. Materials and Methods • Contaminated Soil (SNL) • Sandy loam with low organic carbon (0.8 %) • Doped with ~ 2,700 mg/kg TNT • Doped with ~ 1,000 mg/kg RDX • Spinacia oleracea • Obtained fresh from supermarket as needed • Preparation procedures (Medina et al.2002) • Puree grinding with extract cocktail

  13. Materials and Methods • Extraction Cocktail (Nakagawa et al. 1985) • Buffered Protease Inhibitor • Isopropyl Alcohol • Phenylmethylsulfonyl Fluoride • Protease inhibitor • Ethylenediaminetetraacetic Acid • Metalloprotease inhibitor • DL-Dithiothreitol • Reduces disulfide (-SH) bonds • Potassium Phosphate • pH 8.0

  14. Materials and Methods • TNT Analysis (EPA Method 8515) • Base acetone extraction • KOH and Na2SO3 • Color development • Jackson-Meisenheimer reddish-pink complex • Measure absorbance • Spectrophotometer at 540 nm • Use of a control and blank correction

  15. Materials and Methods • Nitrate Reductase Activity • Harley (1993) Colorimetric Method • Sulfanilamide (SA) • N-1-naphthylethylenediamine-HCL (NEED) • React 10 minutes and read absorbance (540 nm) • Protein Analysis • Bradford (1976) Assay • Bovine serum albumin (BSA) as standard • Coomassie dye-binding reagent • React 10 minutes and read absorbance (595 nm)

  16. Materials and Methods • General TNT Transformation Procedure • Mix sample with crude extract and NADH assay • React for specific time and at fixed temperature • Extract duplicate samples with acetone • Filter sample (0.45mm filter) • Add color developer reagents • Read absorbance @ 540 nm • Run a side-by-side control and correct for blank

  17. Materials and Methods • Aqueous Phase Microcosms • 20 mg/L TNT • 2, 5, 10, 17.5, and 25 g/100 ml Spinacia oleracea • 5, 10, 20 and 30 oC • Soil-slurry Phase Microcosms • ~ 2,700 mg/kg TNT with ~ 1,000 mg/kg RDX • 1 g soil in 25 mL reagent water • 5, 10, 15, 20, and 25 g/100 ml Spinacia oleracea • 5, 10, 20, and 30 oC

  18. Materials and Methods • Unsaturated Soil Microcosms • ~ 2,700 mg/kg TNT with ~ 1,000 mg/kg RDX • 250 g soil per microcosm at 20 oC • 5, 15, and 25 g/100ml Spinacia oleracea • Duplicate 1 g samples extracted every third day • Moisture level evaluated daily • Crude enzyme re-applied every third day

  19. Proposed Kinetic Model • 2nd Order Rate of TNT Degradation (r) • C = TNT concentration (mg/L) • A = enzyme activity (U/L) • ka = 2nd order rate constant (hr-1/U/L) • k = 1st order rate constant (hr-1)

  20. Proposed Kinetic Model • Under Excess Enzyme Activity (1st order) • With Possible Rate Saturation

  21. Results and Discussion • Measured Method Detection Limits • TNT in Water • 0.18 mg/L (2 concentrations with 7 samples each) • TNT in Soil • 4.3 mg/kg (1 concentration and 7 samples) • Nitrate Reductase Activity (mol/min) • Correlated with initial spinach used (g/100 mL) • Linear relationship observed (r2 ~ 1))

  22. Results and Discussion • Aqueous Phase

  23. Results and Discussion

  24. Results and Discussion • Aqueous Phase Results at 20 oC

  25. Results and Discussion • Aqueous Phase Transformation at 20 oC • Pseudo 1st order kinetics observed • k depends upon initial spinach used (g/100 mL) • k decreased as spinach concentration decreased • Enzyme saturation effect indirectly observed • k normalized to applied enzyme activity followed the proposed rectangular hyperbolic kinetic model • Woolf-Hanes linear transform of data allowed for calculation of kmax and Ksat

  26. Results and Discussion

  27. Results and Discussion

  28. Results and Discussion

  29. Results and Discussion • Aqueous Phase Results at T oC

  30. Results and Discussion • Aqueous Phase Transformation at T oC • k decreased as temperature decreased • Range 5 to 30 oC • k followed an Arrhenius relationship • Estimated activation energy 54.7 kJ/mol • Medina et al. (2000) ~ 62.3 kJ/mol • TNT and Myriophyllum aquaticum (parrotfeather)

  31. Results and Discussion • Soil-slurry Phase

  32. Results and Discussion

  33. Results and Discussion • Soil-slurry Phase Results at 20 oC

  34. Results and Discussion • Soil-slurry Phase Transformation at 20 oC • Pseudo 1st order kinetics observed • k decreased as initial spinach used decreased • k an order of magnitude lower than aqueous phase • Enzyme saturation effect indirectly observed • k normalized to applied enzyme activity followed the proposed rectangular hyperbolic kinetic model • Woolf-Hanes linear transform of data allowed for calculation of kmax and Ksat

  35. Results and Discussion

  36. Results and Discussion

  37. Results and Discussion • Soil-slurry Phase Results at T oC

  38. Results and Discussion • Soil-slurry Phase Transformation at T oC • k decreased as temperature decreased • Range 5 to 30 oC • k followed an Arrhenius relationship • Estimated activation energy 26.1 kJ/mol • Activation energy less than 42 kJ/mol tend to indicate diffusion-controlled reactions (Evangelou 1998)

  39. Results and Discussion

  40. Results and Discussion • Influence of Solution Ionic Strength • Aqueous phase (reagent water) versus aqueous phase (soil water) at 20 oC • Observed ~ 15 % reduction in k rate constant • Binding of charged substrates to enzymes and the movement of charged groups within the catalytic 'active' site are influenced by the ionic composition of the medium (Chaplin 2002 ) • However, no difference in degradation when the k’s were normalized for initial applied enzyme activity

  41. Results and Discussion

  42. Results and Discussion • Influence of Protein Adsorption to Soil • 0, 0.5, 1, and 2 g clean soil in 20 mL water • Crude enzyme from 25 g/100 ml spinach • Bradford assay conducted at 0 and 6 hrs • Duplicate samples • Minimal difference in protein content after 6 hrs with increased soil loading • Slight decrease at highest soil concentration (2 g)

  43. Results and Discussion • Enzyme Inhibition by Soil • 5, 10, 20, 30, 40, and 50 mg/L TNT in 25 mL • Aqueous phase microcosm • Soil-slurry phase microcosm with 1 g clean soil • 10 mL crude enzyme at 25 g/100 mL • TNT removal evaluated after 2 hrs contact • Velocity of reaction (mg/L/hr) determined • Classical Michaelis-Menten kinetics applied

  44. Results and Discussion

  45. Results and Discussion

  46. Results and Discussion • Enzyme Inhibition by Soil (cont’d) • Michaelis-Menten constants via a Woolf-Hanes linear transform • Vmax = 23.2 mg/L/hr (water) • Vmax = 7.3 mg/L/hr (soil) • Km = 50.0 mg/L (water) • Km = 20.6 mg/L (soil)

  47. Results and Discussion • Enzyme Inhibition by Soil (cont’d) • Woolf-Hanes plot • Trend towards equal y-intercepts (uncompetitive) • An uncompetitive inhibitor does not bind with the free enzyme (Evangelou 1998) • Protein sorption experiment results • Lineweaver-Burk and Eadie-Hofstee plots • Provide a similar conclusion (uncompetitive)

  48. Results and Discussion • Unsaturated Soil-slurry Phase

  49. Results and Discussion

  50. Results and Discussion