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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. Introduction Research Focus and Objectives Materials and Methods - PowerPoint PPT Presentation
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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
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
Research Focus and Objectives
• Experimental Approach– Evaluate overall TNT transformation using
• Aqueous phase microcosms
• Soil-slurry phase microcosms
• Unsaturated soil microcosms
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
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
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
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
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)
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 (0.45m 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
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))
Results and Discussion
• 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
Results and Discussion
• 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
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
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
)
Results and Discussion
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)
Results and Discussion
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
Results and Discussion
• Aqueous Phase Results at T oCRun Temperature
(oC)Spinach
(g/100 ml)Loading
(g/g)Activity (U/mL)
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
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)
Results and Discussion
• Soil-slurry Phase
Results and Discussion
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
Results and Discussion
• Soil-slurry 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 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
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
Results and Discussion
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
Results and Discussion
• Soil-slurry Phase Results at T oCRun Temperature
(oC)Spinach
(g/100 ml)Loading
(g/g)Activity (U/mL)
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
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)
Results and Discussion
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
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
Results and Discussion
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
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)
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
Results and Discussion
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
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)
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)
Results and Discussion
• 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)
Results and Discussion
• 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
– 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 Discussion
• 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
Results and Discussion
• 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
Results and Discussion
• 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
Results and Discussion
• 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
Results and Discussion
• Transformation comparison over 15 days
Spinach concentration
(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
Results and DiscussionMoisture Tension
0
5
10
15
20
25
30
0 3 6 9 12 15
Time (d)
Ten
sion
(k
Pa)
25g/100mL DI
25g/100mL
Results and Discussion
• 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
Results and Discussion
• 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)
Results and Discussion
• Comparison of Transformation
Spinach concentration
(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
Results and DiscussionMoisture Tension
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)
Results and Discussion
• 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
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