Rhizosphere-Enhanced Treatment of PAHs at Cold, Remote Locations;

Challenges of Application and Monitoring

Mike Reynolds, Larry Perry, Karen Foley, Dave Ringelberg, Brent Koenen, and Kevin McCarthy

Federal Cold Region Sites Low Input Systems

Low Input SystemsHigh Input Systems

Natural attenuation

IncinerationDig and haul

LandfarmingAir sparging and bioventing

CompostingBioslurry reactors

Low-temperature thermal desorptionSoil washing

COST

Phytoremediation / Rhizosphere enhancement

T I M E

Ground Water

Saturated Zone

Relatively constant conditions

Source Contamination

~~ Mixing ! Natural Attenuation

Water Soluble Contaminants

Relatively constant temperature and moisture

Surface soil is less physically mixed than GW, and undergoes temperature, moisture, carbon and nutrient changes

PermafrostActive Zone

Wide-scale, low-cost, in-situ treatment� Requires less input� Requires fewer specifics about a

site

Problem� Remote locations� Hundreds of sites� Cleanup is costly

� Mobilization & demobilization� Limited alternatives� Short season� Relatively slow activity

Problem� Remote locations� Hundreds of sites� Cleanup is costly

� Mobilization & demobilization� Limited alternatives� Short season� Relatively slow activity

Limitations� Temperature� E-S proximity, MT limitations� Microorganisms (numbers,

diversity, activity, function)� Nutrient & contaminant

availability, bioavailable C� Aeration

Limitations� Temperature� E-S proximity, MT limitations� Microorganisms (numbers,

diversity, activity, function)� Nutrient & contaminant

availability, bioavailable C� Aeration

Time

Rel

ativ

e C

once

ntra

tion

of C

onta

min

ant (

% o

f Co)

0

20

40

60

80

100

Typical Bioremediation

Ideal Bioremediation

Impact of Limitations

Lag may be longer

Residual may be higher

Slope may be shallower

Microbial activity in soil is not constant �but starts and stops many timesf (temperature, moisture, carbon additions, ???)

� Analog enrichment (natural forced molecular evolution)

� Stimulated microbial #s & activity

� Reduced M-T limitations, pseudo-mixing

� Carbon-enriched environment

� Not necessarily plant uptake

� Increased OM - Humification

� ???

Stimulating Microbial Activity via the

Rhizosphere Effect

CO2CO2

CO2CO2

CO2

Rhizosphere Fallow

Variability in Contaminant and Productconcentrations makes their routine use for monitoring difficult, and this is exacerbated for:

� Surface soils� Non �brute-force� treatment

methods� Most situations � whether

implementation, regulatory monitoring, or research studies

Reynolds, C. M. 1993. Field measured bioremediation rates in a cold region landfarm: Spatial variability relationships.

Landfarm at Fairbanks Airport� Tilled ~ weekly� Fertilized and limed� Irrigated� Periodically, composite samples

taken near 25 nodes� Calculated half-lives varied ~7-X

Measuring Contaminant Loss is Difficult

Fairbanks Site

ReplicatedGrasses, Nutrients, Both, Control�Soil sock� approachCrude and Diesel

Vegetated

The rhizosphere�effect really does work � Fairbanks data

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Time (min.)

T=1

Fall 1995

T=2

Spring 1996

T=3

Fall 1996

T=4Spring 1997

T=5Fall 1997

T=0

Summer 1995

T=5

Fall 1997

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Time (min.)

T=5

Fall 1997

T=1

Fall 1995

T=2

Spring 1996

T=3

Fall 1996

T=0

Summer 1995

T=4

Spring 1997

~700 days

TPH Changes

Days0 28 56 84 112 140 168 196

TPH

(mg/

kg)

0

1000

2000

3000

4000

5000

6000

Vegetated

SterileUnvegetated

Lab studyAlaska soilWinter ryeNo nutrients added

Significant plant effect

Hexadecane

Days0 28 56 84 112 140 168 196

0

200

400

600

800

Sterile

Unvegetated

Vegetated

Easy to degrade,Little difference

Pyrene

Days0 28 56 84 112 140 168 196

Pyre

ne (m

g/kg

)

0

200

400

600

800

Vegetated

UnvegetatedSterile

Recalcitrant, big difference

Selected CompoundsNon-vegetated

Selected CompoundsVegetated

Selected CompoundsNon-vegetated

We can subtract Vegetated from Non-Vegetated to visual rhizosphere effect

Benefit due to Rhizosphere Effect [ Non-veg ] - [ Vegetated ] is f (Compound and Time)

Rhizosphere effect has Time and Compound Component s

Petroleum - a Complex Mix of hydrocarbons

AsphaltenesPolars

AromaticsPAH

Saturates

Volatiles

Compositional Variability� May consist of 100s to 1000s of

hydrocarbons� Natural or remediation-enhanced weathering

of hydrocarbons in soil substantially alters its composition

� Chromatographic methods yield most accurate measures of extractable hydrocarbons

� Inherent variability in TPH can be minimized by normalization to a recalcitrant marker compound

Generalized Composition of Crude Oil

Biomarkers�

Benefits to Normalizing TPH to Hopane

� Precision (concentration data) can be increased when data are normalized to internal marker such as hopane

� Raw TPH soil data has inherently high variability

� Concentration data can become more variable as petroleum weathers

� Normalizes TPH concentration data

0102030405060708090

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Months

Depl

etio

n of

Tot

al H

ydro

carb

ons

Raw TPH data

NormalizedTPH data

Assumes that contaminant at a site has uniform composition at t=0

Biomarkers�

In addition to TPH, we can normalize other compounds w.r.t a biomarker

Two Field Demo Sites in Korea

Not cold sites, but in practice similar problems

to cold regions

FactorialRyegrass (Yes / No)Nutrients (Yes / No

4 repsComposite samples

Monitoring Rhizosphere-enhanced remediation�

� Petroleum� 1000s of compounds� biodegrade at different rates� by different enzymes

� We can exploit differential rates� confirm degradation� compare treatments

� And it may also give us better insight

C P F P+F

KoreaSite 2

� These data further demonstrate rhizosphere-enhancement in field

Benefit due to Rhizosphere Effectis f (Compound and Time)

Lab Studies

Time

Recalc

itrance

�Response� or Control -

Treatment

$ Data also suggest that for effective monitoring, you must know:� How to sample� What to look for � When to look for it�

� and when is f (microbial processes), not our calendars

� � and microbial processes are f (conditions)�{H2O, Temp., Carbon additions}

How can we do this?

Campion

FactorialVegetation (Yes / No)Nutrients (Yes / No

4 repsComposite and soil sock samples

Campion

� �Bottom� of bioventing pile� Previously fertilized and treated

-350%-300%-250%-200%-150%-100%

-50%0%

50%100%150%200%

C F F+P P

Campion, TPH DepletionsCampion � TPH Depletions

Campion, t1-t2, PAH Depletions

-600%-500%-400%-300%-200%-100%

0%100%200%300%

C F F+P P

Campion � PAH Depletions

� Weathered at t=0

� �Polishing�� Most effect is

with PAHs

Annette Islandactorial

Vegetation (Yes / No)Nutrients (Yes / No

repsomposite and soil sock samples

Annette Island

Annette TPH Depletions

-350%-300%-250%-200%-150%-100%

-50%0%

50%100%150%

C F F+P P

Annette Island � TPH Depletions

C PF+PF

Annette % PAH depletion t1 to t4by treatment

-400%

-300%

-200%

-100%

0%

100%

200%

300%

C F F+P P

Annette Island � PAH Depletions

� Non-uniform composition

� Nutrients and (Nutrients and Plants) starting to respond

Barrow

Barrow

Barrow, t1-t4, %TPH Depletion

-80%-60%-40%-20%

0%20%40%60%

C F PF+P

Barrow - TPH Depletions

Barrow, t1- t4, %PAH Depletion

-20%

0%

20%

40%

60%

80%

C F F+P P

Barrow � PAH Depletions

Non-Freezing Degree Days

010002000300040005000600070008000

Campion Annette Island Barrow

-350%-300%-250%-200%-150%-100%

-50%0%

50%100%150%200%

C F F+P P

Campion, TPH DepletionsCampion � TPH Depletions

-350%-300%-250%-200%-150%-100%

-50%0%

50%100%150%200%

C F F+P P

Campion, TPH DepletionsCampion � TPH Depletions

Campion, t1-t2, PAH Depletions

-600%-500%-400%-300%-200%-100%

0%100%200%300%

C F F+P P

Campion � PAH DepletionsCampion, t1-t2, PAH Depletions

-600%-500%-400%-300%-200%-100%

0%100%200%300%

C F F+P P

Campion � PAH Depletions

Annette TPH Depletions

-350%-300%-250%-200%-150%-100%

-50%0%

50%100%150%

C F F+P P

Annette Island � TPH DepletionsAnnette TPH Depletions

-350%-300%-250%-200%-150%-100%

-50%0%

50%100%150%

C F F+P P

Annette Island � TPH Depletions

Annette % PAH depletion t1 to t4by treatment

-400%

-300%

-200%

-100%

0%100%

200%

300%

C F F+P P

Annette Island � PAH Depletions

Annette % PAH depletion t1 to t4by treatment

-400%

-300%

-200%

-100%

0%100%

200%

300%

C F F+P P

Annette Island � PAH Depletions

C PF+PF

Barrow, t1-t4, %TPH Depletion

-80%-60%-40%-20%

0%20%40%60%

C F PF+P

Barrow - TPH DepletionsBarrow, t1-t4, %TPH Depletion

-80%-60%-40%-20%

0%20%40%60%

C F PF+P

Barrow - TPH Depletions

Barrow, t1- t4, %PAH Depletion

-20%

0%

20%

40%

60%

80%

C F F+P P

Barrow � PAH Depletions

Barrow, t1- t4, %PAH Depletion

-20%

0%

20%

40%

60%

80%

C F F+P P

Barrow � PAH Depletions

Vegetated

Rhizosphere effect improves degradation�and

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T=1

Fall 1995

T=2

Spring 1996

T=3

Fall 1996

T=4Spring 1997

T=5Fall 1997

T=0

Summer 1995

T=5

Fall 1997

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T=5

Fall 1997

T=1

Fall 1995

T=2

Spring 1996

T=3

Fall 1996

T=0

Summer 1995

T=4

Spring 1997

~700 days

Control diesel Spring 96

p.putida51%R.erthro

42%

unknown5%

M agilis2%

Control diesel Fall 96

p.putida52%

R.erthro18%

unknown14%

M.varians12%

B.cepacia4%

Control diesel Spring 97

p.putida45%

unknown21%

B.cepacia19%

R.erthro4%

A.ilicis4%

b.pumilis3%

S.prot3%

y.pseud1%

Veg/Fert diesel Spring 97

M varians18%

no match b15%

p.putida13%A.ilicis

9%

P.shigelloides6%

C.flaccumfaciens6%

d unknowns6%

match f5%

o match g5%

no match h5%

S.spiritivorum4%

R.aquatilis2%

M.mesophilicum2%

B.pumilus2%

R.fascians1%cyto.johnsonae

1%

Veg/Fert diesel Fall 96

p.putida53%

M.varians17%

Sphingobacterium (spiritivorum)

15%

Cytophaga (johnsonae)9%

unknow n4%

Curt. flacc2%

Veg/Fert diesel Spring 96

p.putida87%

S. malto8%

unknown5%

CulturableBacterialDiversity

Contaminant decreases are related to microbial shifts

Culturable

Selective Media & FAME" CFUs/g" Identification of

culturable organisms

How can we characterize microorganisms in soil???

Phenotype

PLFA (Phospholipid fatty acids)

" Biomass" Community Composition" Physiological Status

Genotype

PCR (Polymerase Chain Reaction)t-RFLP (Terminal Restriction Fragment Length

Polymorphism) TGGE and DGGE (temperature/density gradient gel

electrophoresis)RT-PCR (real-time or quantitative PCR)

Enzyme

" Almost an expression of activity - Use DNA fragments for in situestimate of activity

" Soon � use RNA fragments for in situestimate of gene expression! enzyme synthesis

Summary� It�s a system� We can adjust fertilizer and select plant species� Water and temperature more difficult to alter

Monitoring �??� �Standard� methods may not be appropriate� Multiple lines of evidence

� More �selective� chemical techniques� Possibly microbial techniques?

� The best response variable for monitoring may change with �status� of the system

� Phyto� Generally ≅ fertilizer initially� Phyto > fertilizer as recalcitrance ↑

� Fits well with concept of root-released carbon