NovelNovel ApproachApproach toto MeasurementMeasurement ofof RhizosphereRhizosphere EffectEffect inin PhytoremediationPhytoremediation

G. Thoma, T. Lam, S. Ziegler and D. Wolf University of Arkansas, Fayetteville Phytoremediation: The use of plants and their associated microbiota, soil amendments, and agronomic techniques to remove or render harmless environmental contaminants. (Cunningham et al., 1996) Rhizodegradation

¾¾CarbonCarbon enrichedenriched environmentenvironment ¾Exudates/lysates ¾¾HigherHigher microbialmicrobial #s#s andand activityactivity ¾¾RootRoot ‘exploration’‘exploration’ ofof thethe soilsoil

Rhizosphere interactions stimulate contaminant degradation by enhancing soil physical, chemical, and biological properties (adapted from Reynolds et al., 1999) Conceptual Mathematical Model

Root Root Root growth senescence

Bulk Soil Root centric Decay rhizosphere layers root

Root decomposition

Volume and concentration variations: 18 differential equations SensitivitySensitivity AnalysisAnalysis ofof MathematicalMathematical ModelModel

1

0.9 1 0.8 2 3

0.7 4 5 0.6 1 no bulk degradation; annual species; 0.6 mm rhizosphere

Mass Fraction Remaining 2 bulk degradation; perennial species; 0.6 mm rhizosphere 0.5 3 bulk degradation; annual species; 0.6 mm rhizosphere 4 bulk degradation; annual species; 0.6 mm rhizosphere; 2x rate 5 bulk degradation; annual species; 1.5 mm rhizosphere 0.4 0 500 1000 1500 2000 Time (days)

„ The rhizosphere degradation rate constant is kept constant at a value of 0.001 day-1 (Schwab et. al., 1995) with the exception of curve 4 where it was given a value of 0.002 day-1. The average root volume is 0.2% of the total soil volume (White et al., 2001). Pyrene Degrader Population 62 Days After Planting

a b Roxana fine sandy loam c Alfalfa, bermudagrass, soil

fescue, y 8

annual ryegrass, dr plant species mixture, g 6 ddd non-vegetated

-1 MPN/ 4

Pyrene at 500 mg kg g

Greenhouse: 12/12 h; Lo 2 25 + 3oC Harvested 48, 55, 62, 0 ----Contaminated------Uncontaminated---- and 69 d

d

d

e

e

t

t

k

k

a

a

l

l

t

t

u

u

e

e

g

g Bulk B Bulk

B Non- Non-

e

e

V Vegetated

V Vegetated Rhizosphere Rhizosphere PyrenePyrene concentrationsconcentrations soilsoil 6262 daysdays afterafter plantingplanting Treatment Vegetated bulk Rhizosphere (mg kg-1) Alfalfa 74 19 Bermudagrass 48 6 Fescue 141 229 Ryegrass 46 88 Plant Species Mix 53 94

Non-Vegetated Bulk 99 99 CharacterizationCharacterization ofof RhizosphereRhizosphere PropertiesProperties

„ InvestigatedInvestigated properties:properties: „ Microorganism population, root exudates, water, nutrients, contaminant degradation. „ DestructiveDestructive methods:methods: „ Remove grass „ Gently shake to remove bulk soil „ Adhering soil defined as the rhizosphere „ May not be completely representative of rhizosphere soil „ NonNon--destructivedestructive methods:methods: „ Dye indicator in agar plate coupled with optical densitometer (Ruiz and Arvieu, 1990): artificial environment „ Computer tomography and nuclear magnetic resonance. „ SR-FTIR spectromicroscopic system (Raab and Martin, 2001): expensive, scattering of IR in soil matrix. ObjectivesObjectives andand HypothesesHypotheses

„ Objectives:Objectives: „ Determine in-situ rhizosphere thickness. „ Determine in-situ PAH degradation rate. „ Hypotheses:Hypotheses: „ Contaminant depletion in soil zone adjacent to the root surface will be visible. Anticipated „ Differences in luminescent intensity of depletion of PAHs under UV light will be observed phenanthrene in the between rhizosphere and bulk soil and over rhizosphere time. MaterialsMaterials andand MethodsMethods Soybean „ Deposit a thin PAH film onto a quartz plate.

„ Assemble a inclined rhizotron with the quartz Root plate on one side. Pyrene „ Grow plants under controlled environment in a BOD chamber. Quartz plate „ Photograph PAH film „ Digitize and perform Mini-Rhizotron luminescent intensity analysis MaterialsMaterials andand MethodsMethods (cont.) Dry ice & Quartz acetone plate „ PAHPAH filmfilm preparationpreparation

„ Physical vapor deposition Carbon „ P = 1.9 torr. filter „ Uniform fine structure film.

„ UniformUniform heatheat distributiondistribution

„ Aluminum chamber.

„ Thick bottom plate and insulated wall (1500C).

„ ColdCold substratesubstrate (quartz)(quartz) 0 „ Dry ice and acetone (-78 C). To vacuum Insulated pump Chamber „ Minimizes surface diffusion. PAH Deposition Chamber MaterialsMaterials andand MethodsMethods (cont.)

„ Image Acquisition

„ Camera: Nikon 8008s w/ Nikkor 60mm (macro)

„ UV lamp: 100W; fixed relative position

„ Bellows: Nikon PB-6E (22X magnification)

„ XY positioning platform

„ Scanner: Minolta Dimage Scan Elite 5400

„ 4000 dpi (20 Mpixels) ImageImage CalibrationCalibration andand RhizosphereRhizosphere ThicknessThickness CalculationCalculation

Image „ Digital color image is converted acquisition into binary image for simpler Calibrated calculation algorithm. microscope „ Cutoff range for the rhizosphere region is determined by algorithm. 180 μm „ Properties such as area, length Image and width are measured. Image analysis analysis

257 pixels

Pixel size: 530 pixels Rhizosphere 0.7 μm Thickness SoybeanSoybean onon PhenanthrenePhenanthrene (3(3 weeks)weeks)

Visible light illumination Ultraviolet light illumination SoybeanSoybean onon PhenanthrenePhenanthrene (4(4 weeks)weeks) CloseClose--upup VisualizationVisualization ofof Rhizosphere:Rhizosphere: SoybeanSoybean onon PhenanthrenePhenanthrene

10 days 2.5 mm 21 days 28 days DevelopmentDevelopment ofof BermudaBermuda RhizosphereRhizosphere onon PyrenePyrene

30 days 32 days

35 days 42 days RootRoot andand RhizosphereRhizosphere DimensionDimension

RhizosphereRhizosphere RootRoot ThicknessThickness diameterdiameter ((μμm)m) ((μμm)m) SoybeanSoybean ++ phenanthrenephenanthrene 370370 400400

BermudaBermuda ++ 280280 140140 pyrenepyrene

Rhizosphere thickness defined as the zone of clearing QuantitativeQuantitative ImageImage Analysis:Analysis: PyrenePyrene FilmFilm UniformityUniformity andand LuminescentLuminescent IntensityIntensity CalibrationCalibration

„ Deposit pyrene thin films with different deposition time. „ Take UV illuminated photographs at 9 positions on the deposited pyrene thin film. 1 2 3 „ Calculate of the image’s intensity with Matlab program. 6 5 4 „ Calculate of mean and standard 7 8 9 deviation of the intensity. „ Dissolve the pyrene film with acetone and analyze pyrene concentration or mass with HPLC MassMass ofof PyrenePyrene andand DepositionDeposition TimeTime CorrelationCorrelation 1.2 ) 2 1.0 g/mm

μ 0.8

0.6

0.4

0.2 Mass of pyrene ( 0.0 0 20406080100 Deposition time (min) LuminescentLuminescent IntensityIntensity vs.vs. PyrenePyrene MassMass

18000 16000 14000 12000 10000 8000 Intensity y = -47299x2 + 55651x - 220.3 6000 R2 = 0.9972 4000 2000 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Mass of pyrene (μg/mm2) RhizosphereRhizosphere ContaminantContaminant DegradationDegradation RateRate

Crop Area #3 Crop 1945 x 3690 Area #1 562 x 786

Crop Crop Area #4: Area #2 503 x 685 454 x 1608

„ Photograph rhizosphere over time.

„ Crop the image at different positions.

„ Calculate the crop image luminescent intensity.

„ Calculate mass of pyrene remaining.

„ Calculate degradation rates BermudaBermuda RhizodegradationRhizodegradation RateRate ConstantConstant

30 days 32 days

35 days 42 days DegradationDegradation Rate:Rate: ZoneZone 11

0.0

-0.1

-0.2 y = -0.0251x + 0.4316 -0.3 R2 = 0.6952 -0.4 Ln(m/m0) -0.5

-0.6

-0.7 30 32 34 36 38 40 42 44 Time (days) DegradationDegradation Rate:Rate: ZoneZone 22

0.0 -0.2 -0.4 -0.6 ) 0 -0.8 -1.0 Ln(m/m -1.2 -1.4 y = -0.0739x + 1.5049 -1.6 R2 = 0.9643 -1.8 30 32 34 36 38 40 42 44 Time (days) DegradationDegradation Rate:Rate: ZoneZone 33 -- EntireEntire RhizosphereRhizosphere

0.0 -0.1 -0.2 y = -0.0341x + 0.6315 -0.3 R2 = 0.9112 -0.4 -0.5

Ln(m/m0) -0.6 -0.7 -0.8 -0.9 30 35 40 45 Time (days) ConclusionsConclusions

„ NonNon--destructive,destructive, nonnon--invasiveinvasive quantificationquantification ofof rhizosphererhizosphere thicknessthickness andand inin--situsitu PAHPAH degradationdegradation raterate areare possible.possible. „ RhizosphereRhizosphere thicknessthickness andand contaminantcontaminant degradation:degradation: „ ExpandExpand overover time.time. „ VaryVary alongalong thethe root.root. „ DependsDepends onon plantplant type,type, plantplant growthgrowth condition,condition, soilsoil structurestructure andand otherother factors.factors. „ UnderstandingUnderstanding contaminantcontaminant dynamicsdynamics inin rhizosphererhizosphere isis neededneeded forfor modelingmodeling efforts.efforts. AcknowledgementAcknowledgement

„ ThisThis projectproject isis fundedfunded byby thethe EPAEPA throughthrough thethe IntegratedIntegrated PetroleumPetroleum EnvironmentalEnvironmental Consortium.Consortium.

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