Clint Richardson and Enric Bonmati

Phytoremediation of TNT Contaminated Soils using Nitrate Reductase Enzyme Extract from

Spinacia oleracea

Clint Richardson and Enric Bonmati

Department of Civil and Environmental EngineeringNew Mexico Tech

Introduction

Research Focus and Objectives

Materials and Methods

Proposed Kinetic Model

Results and Discussion

Conclusions

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)

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

Introduction

• TNT Molecular Structure (C5H7N3O6)

Ref. McFarlan (2000)

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)

Introduction

• TNT Oxidation/Reduction Transformation

Ref. McFarlan (2002)

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

• Spinacia oleracea– High content of nitrate reductase enzyme

• Capable of interacting with NACs

– Commercially available or easily grown


• 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


• Experimental Approach– Evaluate overall TNT transformation using

• Aqueous phase microcosms

• Soil-slurry phase microcosms

• Unsaturated soil microcosms


• 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


• 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

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


• 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


• 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)


• 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 (0.45m filter)– Add color developer reagents – Read absorbance @ 540 nm– Run a side-by-side control and correct for blank

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

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

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)

kCCAkdt

dCr a

Proposed Kinetic Model• Under Excess Enzyme Activity (1st order)

• With Possible Rate Saturation

AKkk

eC

C

sat

kt

o

1max


• 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))


• Aqueous Phase

Results and DiscussionAqueous Phase Transformationversus Spinach Concentration

0.0

0.2

0.4

0.6

0.8

1.0

0 2 4 6 8

Time (hrs)

C/C

o

25 g/100 mL17.5 g/100 mL10 g/100 mL5 g/100 mL2 g/100 mL


• Aqueous Phase Results at 20 oCRun Temperature

(oC)Spinach

(g/100 ml)Loading

(g/g)Activity (U/mL)

k (hr-1)

ka

(hr-1/U/mL)

1 20 25 5 2.06 0.49 0.24

2 20 17.5 3.5 1.78 0.43 0.24

3 20 10 2 0.79 0.39 0.49

4 20 5 1 0.27 0.30 1.10

5 20 2 0.4 0.08 0.24 3.00


• 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

Results and DiscussionAqueous Phase Rectangular Hyperbola Kinetics

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0 0.5 1 1.5 2 2.5

Activity (U/mL)

k (

hr-1

)


Alternate View of Previous Kinetics

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.000 0.001 0.002 0.003 0.004 0.005

Spinach Mass/TNT Loading (g/ug)

k (

hr-1)

10 mg/L20 mg/L40 mg/LMedina et al. (2000)


Aqueous Phase Kinetics at 25 g/100 mL

y = e-0.11x

y = e-0.26x

y = e-0.48x

y = e-0.89x

0.0

0.2

0.4

0.6

0.8

1.0

0 4 8 12 16 20Time (hrs)

C/C

o

5 C10 C20 C30 C


• Aqueous Phase Results at T oCRun Temperature

(oC)Spinach

(g/100 ml)Loading


k (hr-1)

ka

(hr-1/U/mL)

1 5 25 5 1.60 0.11 0.07

2 10 25 5 1.93 0.26 0.13

3 20 25 5 2.06 0.49 0.24

4 30 25 5 1.79 0.89 0.50


• 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)


• Soil-slurry Phase


Soil-slurry Phase Transformationversus Spinach Concentration

0

0.2

0.4

0.6

0.8

1

0 4 8 12 16 20 24

Time (hrs)

C/C

o

25 g/100 mL

20 g/100 mL

15 g/100 mL

10 g/100 mL

5 g/100 mL


• Soil-slurry Phase Results at 20 oCRun Temperature

(oC)Spinach

(g/100 ml)Loading


k (hr-1)

ka

(hr-1/U/mL)

1 20 25 1.25 1.96 0.032 0.016

2 20 20 1 1.86 0.024 0.013

3 20 10 0.75 1.24 0.030 0.024

4 20 5 0.50 1.09 0.027 0.024

5 20 2 0.25 0.66 0.018 0.027


• 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


the proposed rectangular hyperbolic kinetic model

• Woolf-Hanes linear transform of data allowed for calculation of kmax and Ksat


Soil-slurry Phase Rectangular Hyperbola Kinetics

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.0000 0.0005 0.0010 0.0015

Spinach/TNT Mass Loading (g/ug)

k (h

r-1)

Results and DiscussionSoil-slurry Phase Kinetics at 25 g/100 ml

y = e-0.048x

y = e-0.032x

y = e-0.027x

y = e-0.017x

0.0

0.2

0.4

0.6

0.8

1.0

0 4 8 12 16 20 24

Time (hrs)

C/C

o 30 C

20 C

10 C

5 C


• Soil-slurry Phase Results at T oCRun Temperature

(oC)Spinach

(g/100 ml)Loading


k (hr-1)

ka

(hr-1/U/mL)

1 5 25 1.25 2.16 0.017 0.008

2 10 25 1.25 2.02 0.027 0.014

3 20 25 1.25 1.96 0.032 0.016

4 30 25 1.25 2.04 0.048 0.024


• 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)


Arrhenius Temperature Relationships

0.01

0.10

1.00

0.38 0.40 0.42 0.44

Inverse RT (mol/kJ)

k (

hr-1

)

Soil 26.1 kJ/mol r2 = 0.93Water 54.7 kJ/mol r2 = 0.96


• 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


Solution Ionic Strength Effect at 20 oC

y = e-0.41x

y = e-0.48x

0.0

0.2

0.4

0.6

0.8

1.0

0 1 2 3 4 5 6

Time (hrs)

C/C

o

Soil Water

Reagent Water


• 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)


• 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


Michaelis-Menten Kinetics

0

2

4

6

8

10

12

14

0 10 20 30 40 50 60TNT (mg/L)

Vel

ocity

(mg/

L/h

r) Aqueous PhaseSoil-slurry Phase

Results and DiscussionWoolf-Hanes Plot

y = 0.13x + 3.07

r2 = 0.97

y = 0.046x + 2.11

r2 = 0.95

0

2

4

6

8

10

0 10 20 30 40 50 60

TNT (mg/L)

Con

cent

rati

on/V

eloc

ity

(mg/

L/m

g/L

hr) Soil

Water


• 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)


• 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)


• Unsaturated Soil-slurry Phase

Results and DiscussionTNT Degradation over 30 days

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

0 3 6 9 12 15 18 21 24 27 30Time (d)

C/C

o

Control

5g/100mL

15g/100mL

25g/100mL

Results and DiscussionUnsaturated Soil Rectangular Hyperbola Kinetics

0

0.01

0.02

0.03

0.04

0.05

0.06

0.00 0.02 0.04 0.06 0.08 0.10 0.12

Average Activity (U/g)

k (d

-1)


• Unsaturated Soil Transformation at 20 oC– Pseudo-first-order kinetics observed

• Trend to zero-order kinetics after long time• k an order of magnitude lower than soil-slurry phase• k increased with increased spinach loading


the proposed rectangular hyperbolic kinetic model• Woolf-Hanes linear transform of data allowed for

calculation of kmax and Ksat


• Overall Transformation over 30 days

Spinach concentration

(g/100 mL)

Ave. daily rate transformed

(mg/d)

Normalized Ave. daily rate

transformed

(mg/g/d)

TNT transformed

(%)

25 16.1 0.21 77.1

15 14.7 0.31 70.4

5 13.3 0.83 63.7

Results and DiscussionMoisture Tension

0

10

20

30

40

50

60

0 3 6 9 12 15 18 21 24 27 30

Time (d)

Ten

sion

(k

Pa)

Control25g/100mL15g/100mL 5g/100mL


• Transformation for Unsaturated Soil– Normalized average daily rate (mg/d/g)

• Higher rate of transformation observed for microcosms at lower spinach concentration loading

– Better efficiency is achieved at lower applied dosing of enzyme over a longer remediation period

– Percent TNT transformation• Lower overall removal for microcosms at lower

spinach concentration loading


• Transformation for Unsaturated Soil (cont’d)– Percent of TNT transformed over the first 15 days

relative to the total TNT removal was higher for the microcosms with higher spinach concentration loading

• Reflects the higher kinetic rate observed for the stronger applied enzyme solutions

– TNT transformed per mass of spinach used (mg/g) increased for the microcosms at lower spinach concentration loading


• Effect of enzyme application cycle at 20 oC– First microcosm (5 cycles over 15 days)

• Crude extract at 25 g/100 mL spinach concentration

• Reagent water only every alternate cycle

– Second microcosm (5 cycles over 15 days)• Crude extract at 25 g/100 mL spinach concentration

– Moisture drying pattern virtually identical

Results and Discussion Effect of Enzyme Application Frequency

0.50

0.60

0.70

0.80

0.90

1.00

0 3 6 9 12 15

Time (d)

C/C

o

25g/100mL

25g/100mL w DI


• Transformation comparison over 15 days


(g/100 mL)

Ave. daily rate transformed

(mg/d)

Normalized Ave. daily rate transformed

(mg/g/d)

TNT transformed

(%)

25 16.8 0.46 37.1

25 + DI 12.8 0.54 28.3


0

5

10

15

20

25

30

0 3 6 9 12 15

Time (d)

Ten

sion

(k

Pa)

25g/100mL DI

25g/100mL


• Effect of enzyme application cycles (cont’d)– No transformation during reagent water application– Moisture tension for each microcosm over the 15

days of test were similar– Overall percent removal lower for alternating cycle– Higher normalized average daily rate was observed

for the alternate application of enzyme• Better efficiency is again achieved at lower applied

dosing of enzyme over a longer remediation period


• Effect of Moisture on Transformation– Two experiments over 15 days at 20 oC– 25 g per 100 mL spinach concentration– Average applied activity over 15 days

• Approximately equal (24.5 U versus 25.6 U)

– Moisture drying pattern significantly different• k = 0.03 d-1 (wetter) versus k = 0.05 d-1 (drier)

Results and DiscussionComparision of Enzyme Loading

y = e-0.03x

y = e-0.05x

0.40

0.50

0.60

0.70

0.80

0.90

1.00

0 3 6 9 12 15

Time (d)

C/C

o

25g/100mL (36.3 g)25g/100mL (45.0 g)


• Comparison of Transformation


(g/100 mL)

Ave. daily rate

transformed (mg/d)

Normalized Ave. daily

rate transformed

(mg/g/d)

TNT transformed

(%)

Total applied Spinach load

(g)

25 16.8 0.46 37.1 36.3

25 26.0 0.58 62.3 45.0


0

10

20

30

40

50

60

0 3 6 9 12 15

Time (d)

Ten

sion

(k

Pa)

25g/100mL (45.0 g)

25g/100mL (36.3 g)


• Effect of Moisture (cont’d)– Higher normalized average daily rate of

transformation corresponded to the higher loaded microcosm

– Higher loaded microcosm dried out more quickly over each cycle than the lighter loaded microcosm

– Rate constant and overall percent TNT removal was lower in wetter microcosm

Conclusions

• Nitrate reductase activity in pureed Spinacia oleracea can be quantified easily and accurately

• TNT Transformation by nitrate reductase enzyme is adequately described by pseudo-first-order kinetics for constant enzyme activity

Conclusions

• A rectangular hyperbola saturation function may be used to describe transformation kinetics with respect to enzyme activity or applied spinach mass/TNT mass loading– Results in an estimate of a maximum rate of

reaction and a half-saturation constant

Conclusions

• Maximum rate of reaction (kmax)

– aqueous phase 0.50 hr-1

– soil-slurry phase 0.04 hr-1

– unsaturated soil 0.08 d-1

Conclusions

• Half-saturation constants (Ksat*)

– Aqueous phase 0.63 U/mol NO2

– Soil-slurry phase 0.28 U/mol NO2

– Unsaturated soil 0.06 U/g

Conclusions

• Aqueous phase experiments– Transformation kinetics appeared independent

of initial TNT concentration over the range of 10 to 40 mg/L under equivalent applied spinach mass/TNT loading

Conclusions

• Soil-slurry phase experiments– Ionic effects of dissolved species and

adsorption of protein onto soil particles affecting enzyme function were small and not considered to be a significant factor in lower observed kinetics for the stated conditions

Conclusions

• Temperature effects– Aqueous phase and soil-slurry phase

experiments were described by the Arrhenius relationship over the range of 5 to 30 oC

• Estimated activation energies– Soil-slurry phase 26.4 kJ/mol– Aqueous phase 54.7 kJ/mol

Conclusions

• Presence of soil in aqueous phase microcosms– Substantially reduced nitrate reductase efficacy in

the form of uncompetitive inhibition– Additional studies are needed to characterize this

inhibition in terms of the specific inhibitor, inhibitor concentration, and the inhibition constant

Conclusions

• Unsaturated Soil Microcosms– TNT mass to enzyme loading in the unsaturated

soil microcosms was high and kinetics generally slow over the 30 days of study

– Overall transformation of TNT ranged from 63.7 to 77.1 %

Conclusions

• Unsaturated Soil Microcosms (cont’d)– Normalized transformation data (mg/d/g)

suggests that better efficiency may be realized at lower applied dosing of enzyme over a longer remediation period

– Additionally, a longer period in between enzyme application (i.e. dosing frequency) may also be beneficial to improve overall effectiveness of a field protocol

Conclusions

• Unsaturated Soil Microcosms (cont’d)– Moisture level may influence the overall

effectiveness of the nitrate reductase enzyme in an unsaturated soil

– Additional research into this moisture effect is needed to establish this aspect of a workable field protocol

Conclusions

• Future Studies– Based on the general saturation trends observed

and the goodness-of-fit of the transformation data to the proposed function, additional work is needed to delineate the kinetic response of soil microcosms with respect to enzyme activity or applied spinach mass/TNT loading

Conclusions

• Future Studies (cont’d)– Although the TNT transformation products

have been mapped extensively in aquatic plants, additional research is needed to define their kinetics with respect to enzyme activity or applied spinach mass/TNT loading

– TNT intermediates are not quantified using the colorimetric analysis described herein

Acknowledgments

• Waste-management, Education, and Research Consortium (WERC)

• Department of Civil and Environmental Engineering, New Mexico Tech

Documents

Clint Richardson and Enric Bonmati