Clint Richardson and Enric Bonmati

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