Partners: Imperial Oil; Talisman Energy; ConocoPhillips Canada; Northrock Resources; Canetic Resources; BASF; EBA; OAFT; Stantec; Ontario Ginseng Growers, CRA; Region of Waterloo; City of Guelph; NSERC

Successful Field and Lab Tests of a Multi-Process Phytoremediation System for Decontamination of

Petroleum and Salt Impacted SoilsBruce Greenberg, Xiao-Dong Huang, George Dixon,

Perry Gerwing and Bernie Glick

WATERLOO

ENVIRONMENTALBIOTECHNOLOGY Inc

EarthmasterEnvironmental Strategies Inc

Development and Proof of the Multi-Process Phytoremediation System (MPPS)

1. 6 Years of Lab Studies1. Soil spiked with creosote (PAHs)2. Total Petrol. Hydrocarbon (TPH) contam. soil3. PAH contaminated soil from urban foundry4. Plant growth on salt impacted soils

2. 3 Years of Field Studies1. TPH, Talisman Energy, Turner Valley, AB 2005-

062. TPH, North East Alberta 2005-063. TPH, Imperial Oil Land Farm, Sarnia, ON 2004-064. DDT, Simcoe, ON 2005

Advantages of Phytoremediation

1. Improves the natural structure and texture of soil2. It is driven by solar energy and suitable to most

regions and climates3. It is low in cost and technically feasible4. Plants can provide sufficient biomass for rapid

remediation; promote high rhizosphere activity5. Restoration6. > 30,000 sites in Canada where such technology is

needed, > 300,000 sites in the US

Description of the Multi-ProcessPhytoremediation System (MPPS)

Physical soil Till the soil: exposure to sunlight and air treatment: Exposure to sunlight photooxidizes

contaminants

Bioremediation: Inoculation of PAH/TPH degradingbacteria

Phytoremediation: Growth of plants alone on the soil

MPPS: Land farming the soil for two weeksInoculation of PAH degrading bacteriaGrowth of plants with PGPR

PGPR: Plant growth promoting rhizobacteria. Prevents the synthesis of stress ethylene.

Interaction of a PGPR containing ACC deaminase with a plant seed or root

Plant growth promoting rhizobacteria (PGPR)

Two natural, non-pathogenic strains of Pseudomonas putida(UW3 and UW4)

One has high ACC DeaminaseOne is an auxin producer

Both PGPR are applied to seeds prior to planting

Stress Response

Ethylene

ACCSynthase

ACC OxidaseAmmonia and α-ketobutyrate

ACCDeaminase

Plant Tissue

Bacterium

Exudation

Amino Acids

ACC ACC

IAA IAA

SAM

Amino Acids

Cell Elongation and Proliferation

Isolation

Proliferation of selected PGPR

Apply to seed,

Plant seeds

Few days later (Tomato)

Control PGPR Treated

Use of the Multi-Process phytoremediation System (MPPS)

for Removal of Total Petroleum Hydrocarbons from Imperial Oil Soil

(~ 5% TPH)

Greenhouse Studies and demonstration of PGPR effects

Plants only Plants with Degrad. Bacteria

Plants with

PGPR

Multi-Process System

Growth of Tall Fescue – 90 d

Plants only

MPPS

Plants from the MPPS on Land-

Farmed Contaminated Soil

0

20

40

60

80

100

120

140

160

180

200

Root FW Root DW Shoot FW Shoot DW

Plant Biomass

Perc

enta

ge o

f no

cont

amin

ant c

ontr

ol phytoremMulti-system

Plant Biomass for Phytoremediation and the MPPS

Remediation Kinetics of Four Methods Tested

0

10

20

30

40

50

60

0 2 4 6 8

Remediation time, months

THP

rem

aini

ng in

soi

l, g/

kg

landfarmingbioremPhytorem onlyMPPS

Remediation of different fractions of TPHafter 2 4-month seasons

TPH Fractions

0

20

40

60

80

100

120

Fraction 2 Fraction 3A Fraction 3B Fraction 4 Average

Land farming

Phytoremediation

Msystem

TPH

rem

aini

ng, %

con

trol

Contaminants metabolized/degraded: ~40g TPH/kg soil removed, < 100g DW plants/kg soil. Plants cannot be 40 % TPH.

Field Test at Talisman Energy Biopile (1 % TPH)Turner Valley Alberta

Year 1: Summer 2005 – Rye/Fescue Growth

- PGPR + PGPR

+ PGPR

- PGPR

90 dNote: Weather cool and wet. Poor, gravelly soil required fertilizer

Talisman Energy,Turner Valley SiteSummer 2005 Remediation Results

100 d – Year 1

Rye/Fescue+PGPR

Rye/Fescue- PGPR

BLANK

RYE/FESCUE +PGPR

RYE/FESCUE - PGPR

BARLEY/RYE - PGPR

BARLEY/RYE +PGPR

TRITICALE - PGPR

TRITICALE +PGPR

BLANK

~2m ~2m

~70m

35m

10m20m 20m

12 0.58%

13 0.53%

14 0.57%

11 0.52%

15 0.55%

50.73%

60.78%

70.83%

80.87%

4 0. 65%

3 0.48%

31 0.659%

30 0.768%

26 0.609%

24 0.716%

20 0.698%

21 0.751%

9

2 0.55%1 0.76%

10 0.97%

+ PGPR

Avg 0.55%

SE: 0.01%

35 % REM0.89%

Blank

Avg 0.85%

SE: 0.03%

- PGPR

Avg 0.61%

SE: 0.06%

28 % REM

Note: Every plot with PGPR had 15 to 20 % superior remediation

Excellent remediation of recalcitrant contamination on poor soil to a depth of 20 cm in 1st year

CCME Fractions remaining in Turner Valley Soil after 100 d – Year 1

(rye/fescue + PGPR)

0

0.1

0.2

0.3

Blank Plants Blank PlantsCC

ME

Frac

tion,

% D

W o

f Soi

l

Fraction 4Fraction 3

Remediation Kinetics For Turner Valley SoilGreenhouse vs. Field

0

10

20

30

40

50

60

0 2 4 6 8

Remediation time, months

THP

rem

aini

ng in

soi

l, g/

kg

landfarmingbioremPhytorem onlyMPPS

MPPS: Greenhouse 100 days

Field 100 days

Both at 35 to 40 % Remediation in 100 d

~70 m

~70 m

~30 m ~10 m ~30 m

~3 m wide access roads

Rye/Fescue + PGPR

Over seed with new rye fescue + PGPR

Rye/Fescue - PGPR

5 m wide strips for small scale experiments

Blank

Blank

Rye/Fesc + PGPR

Rye/Fesc - PGPR

T/B/A + PGPR

T/B/A - PGPRBarley + PGPR

Barley - PGPR

Site plan May 13th, 2006

Timothy/Brome/Alfalfa(T/B/A) + PGPR

Timothy/Brome/Alfalfa- PGPR

Not to scale

35 m20 m

Sample point

11

1

23

45

6

15

7 8

910

11

12

1314

16

17

19

18

21

22

27

23

2526

24

20

28

32

29

30

33

31

34

35 36

37 38

39

41

40

4255

56

5857

Turner Valley Year 2 2006Planted May 15, 2006

• Quality control• Ryegrass treated with

seed treater• UW3 + UW4 + Me-

Celluose• Test of PGPR efficacy

on 3 % Creosote• Plants grew well in the

field

Control Soil

Control Soil

- PGPR

0.3 % Creosote- PGPR

0.3 % Creosote+ PGPR

Turner Valley, Year 2 2006

• Planted May 15, 2006• Treated with seed treater• UW3 + UW4 + Me-Celluose• Good positive PGPR effect• Excellent growth

June 8, 2006 July 28, 2006

- PGPR + PGPR

Rye Fescue + PGPRRye Fescue

Rye Fescue + PGPR

Sept 5, 2006

Timothy Brome Alfalfa + PGPR

Turner Valley, Year 2 Remediation

0.2

0.4

0.6

0.8

May 15 July 13 Sept 5

Date

TPH

, %

30 % Remediation in 3.5 months

9 % Remediation in 2 months

Same as the remediation kinetics observed in previous years

Turner Valley 2006 – 60 cm deep ploton clay pad - Planted May 15, 2006

• Controlled experiment• Test depth of remediation• UW3 + UW4 + Me-Celluose• Spring ryegrass and tall fescue• Plants have grown well• Positive PGPR effect• Remediation Sept 5:

15 % w/ PGPR, 15 % w/ PGPR, 66 % w/o PGPR% w/o PGPR

June 8, 2006 June 25, 2006

July 28, 2006

- PGPR

+ PGPR + PGPR

- PGPR

- PGPR

+ PGPR

Field Test at Imperial Oil Land Farm, Sarnia, ONYear 1: Summer 2004 – Rye grass

Oil Sludge Total Petroleum Hydrocarbon (TPH) Contaminated Soil (15 % w/w)

MPPS (+ PGPR) Plants alone (- PGPR)

60 after planting

Remediation of TPHs CCME Fractions from Imperial Oil Land

Farm, 120 d

7

14

21

28

F2 F3 F4 >C50

Frac

tion

in so

il, g

/kg

Untreated MPPS

Contaminants Contaminants metabmetaband/or degraded:and/or degraded:

•• ~75 g TPH/kg soil ~75 g TPH/kg soil removedremoved

•• All fractions All fractions removedremoved

•• 100 g DW plants/kg 100 g DW plants/kg soil. soil.

•• Plants cannot be Plants cannot be 75 % TPH.75 % TPH.

Note: Summer 2004 was a cool wet summer

~ 130 m

~ 40 m

10 m5 m

Barley/Rye + PGPR

Barley/Rye - PGPR

Corn - PGPR

Corn + PGPR

F Rye - PGPR

F Rye + PGPR

Fall Rye + PGPR

Blank

Fall

Rye

-PG

PR

10 W

Rye

/Fes

cue

+ PG

PR

25 m

F Rye - Beads

F Ryr + Beads

F Rye – AC1

Fall Rye + PGPR F Rye + AC1

F Rye - PM

F Rye + PM

Triticale - PGPR

Triticale + PGPR

10 mNot to scale

T = Treated with PGPR PM = Peat moss coated with PGPRBeads = Alginate beads containing PGPR

Fall rye + PGPR + PMFall rye + PM

Sarnia Land Farm – 10E and 10 W – 2005 PlantingYear 2

Fall Rye + PGPR + Peat Moss

Fall Rye - PGPR + Peat Moss

Imperial Oil Land Farm, Sarnia, ONYear 2: 2005 – Fall Rye Growth

15 % TPH 60 d 15 % TPH 60 d

10 % TPH 30 d 10 % TPH 90 d

- PGPR + PGPR - PGPR + PGPR

- PGPR+ PGPR+ PGPR

Note: Summer 2005 was unusually hot and dry

Imperial Oil Land Farm, Sarnia, ONSummer 2005 – Rye/Fescue

Results

Root depth (+ PGPR) after 90 d was > 40 cm

Extent of Remediation

+ PGPR30 d 9 % ± 1.560 d 17 % ± 2

120 d 35 % ± 4

- PGPR remediation was about 50 % slower

TPH Removal from Sarnia Year 2

0

6

12

Blank Rye/Fescue

TPH

, % D

W o

f Soi

l

1C

CM

E Fr

actio

n, %

DW

of S

oil 3

1.5

0

Fraction 3

Fraction 4

Blank Plants Blank Plants

Imperial Oil Sarnia Land Farm Year 3

June 19, 2006

+

Rye/Fesc/Barley + PGPR

-+-

Fall Rye overseededwith Rye/Fescue

+ PGPR

Imperial Oil Sarnia Land Farm Year 3 (2006)

• Planted Barley/Fescue/Rye Grass on April 20, 2006• Plants were treated with PGPR (UW3 and UW4)

using a mechanical seed treater

Barley/Rye/Fescue

Rye/Fescue

- PGPR

- PGPR + PGPR

+ PGPR

~ 11 % TPH10 E (~6% TPH)

Barley/Rye/Fescue+PGPR

40 days after planting – Weather good

Imperial Oil Sarnia Land Farm Year 3

60 d after planting60 d after planting

Fall Rye Fall Rye overseededoverseededwith Rye/Fescuewith Rye/Fescue

+ PGPR+ PGPR

100 d after planting100 d after planting

Over seeded Over seeded Rye/Fescue field,Rye/Fescue field,

after fall rye mowedafter fall rye mowed

40 d after planting40 d after planting

100 d after planting100 d after planting

Rye/Fescue,Rye/Fescue,Test plotsTest plots

Remediation data on Aug 15: 13 % ± 3%

Time (min)

0 20 40 60 80

Abs

orba

nce

Uni

ts

0

1

2

3

4

5

Non-remediated soilRemediated soilRoots from rem. soil

Root data rescaled; Qualitatively different HPLC profile

Time (min)0 20 40 60

Abs

orba

nce

Uni

ts

0

1

2

3

4

HPLC assay for TPH uptake by Rye/Fescue roots

~ 35 % of the TPH removed from the soil

Remediated TPH does not appear in the roots

What happened to the TPH?

• TPH removed from the soil, but not in the plant roots

• TPH must have been degraded• Where was it degraded?• Perhaps in rhizosphere by

bacteria, fungi and roots• Soil fungi and bacteria have

very active and diverse metabolic activities

• In soils with PGPR treated plants, bacteria and fungal counts are 5 to 10 fold higher

PGPRSoil bacteriaFungi

TPH

CO2 Acetate

etc

Possible degradation pathway of TPH in Soil

RR'

RR'

O

RR'

OH

OH

RR'

O

O

RR'

O

O

OH HO

[O] P450?

[O]

[O] P450?

H2O

2 Fatty Acids

Aliphatic hydrocarbon

Control Oil consuming bact.Control UW3 (PGPR) Control UW4 (PGPR)

No Inoculation Inoculation w/ PGPR

Grow for 12 h on shaker

SaturatedTPH in Phos-Buff-Saline

PGPR Can Degrade Oil

TPH sole reduced carbon source

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

Microbes in Sarnia Land Farm Soil From Remediation Field Trial 2006

Rye Fescue Barley Soils

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

PGPR Petro Deg Bacteria

Total FungiTotal Bacteria

108

107

106

105

107

106

105

104

103

106

105

104

103

108

107

106

105

104

27.4 27.429.5 29.521.7 21.713.8 13.8

+ Plants + PGPR

+ Plants - PGPR

- Plants - PGPR

Enhancement of Plant Growth on Salt Contaminated Soils

Using PGPR

0

0.5

1

1.5

2

2.5

3

Contro

lNo P

GPR

UW4

UW3UW4+

UW3

Wei

ght (

g)

Barley fresh weight - 10 days on AL

00.5

11.5

22.5

33.5

4

Contro

lNo P

GPR

UW4

UW3UW4+

UW3

Wei

ght (

g)

Shoots biomassRoots biomass

Oat fresh weight - 10 days on AL

Effect of PGPR on Plant growth in Alberta Salt Impacted SoilEC = 2.2 dS/m, SAR = 27, Cl = 260 mg/kg

Oat – 10 d

Control soil No PGPR UW4 UW3 UW4+UW3

Control soil No PGPR UW4 UW3 UW4+UW3

Barley – 10 d

Shoots – 219% increase

Roots – 356% increase

Shoots – 24% increase

Roots – 4.1% increase

Wheat Low 21d Fresh Weight

Promix-PGPR

UW3+UW4Fungus

PGPR+Fungus

Fres

h W

eigh

t (g)

0.0

1.0

2.0

4.0

6.0

8.0

10.0

Oat Low 21d Fresh Weight

Promix-PGPR

UW3+UW4Fungus

PGPR+Fungus

Fres

h W

eigh

t (g)

0.0

2.0

4.0

6.0

8.0

10.0RootShoot

Sask Salt Soil: EC = 14 dS/m , SAR = 11, Cl = 1880 mg/kgOat

Promix -PGPR UW3+UW4 Fungus PGPR+Fungus

7.2g

Shoots and roots 100 % improved3.5g

Wheat

Promix -PGPR UW3+UW4 Fungus PGPR+Fungus

1.1g

0.69g

Shoots and roots 60 to 80 % improved

• MPPS has great potential for efficient remediation of organic, salt and metal contaminated sites

• PGPR is the key: healthy plants with vigorous roots in PAH, TPH, DDT, salt and metal contam. soils

• PGPR alleviate stress and promote growth: Low ethylene and high auxin

• 8 Months in the greenhouse: MPPS removed 90 % of recalcitrant TPHs and PAHs

• 3 years of fields tests successful: MPPS removed 30 % to 60 % of recalcitrant TPHs and DDT per year

• Contaminants metabolized and/or degraded• Great promise for restoration of oil and salt impacted

sites, and brownfields

CONCLUSIONS

• Continued field testing of the MPPS• Increasing research on salt remediation• Ready to deploy at new sites• Proposals for new sites are being

entertained• TPH sites can be remediated in 2 to 4

years • Further research on salt and metal

remediation underway

Future Work

Colleagues and partners• The people that do all

the workKaren GerhardtYola GurskaMark LampiPearl ChangWenxi WangHaitang WangAaron KhalidDavid IsherwoodShan Shan WuJulie NykampAnabel UeckermannXiao-Ming Yu

• CollaboratorsXiao-Dong HuangBernie GlickPerry GerwingGeorge DixonRon Pitblado

• Partners D Bristow, L Lawlor, M Young, Imperial OilJ Gordon, Talisman EnergyA Hopf, BASFG Surgeoner, OAFTG Stephenson, StantecG Brooks, CRAD Hoffman, Ginseng GrowersRegion of WaterlooCity of GuelphNSERC

Phytoremediation of TPH Contaminated Soil Early in Tall Fescue Growth

Plants only

Plants + PAH Deg. bacteria Plants + PGPR MPPS

0

10

20

30

40

50

60

landfarm Biorem phytorem Multi-process Control

Different Remediation Methods

TPH

s re

mai

ning

, g/k

gEffectiveness of Different Methods on TPH Remediation -

Second 4 month season (i.e., 8 months total)

Remediation Kinetics For Land Farm SoilGreenhouse vs. Field

0

10

20

30

40

50

60

0 2 4 6 8

Remediation time, months

THP

rem

aini

ng in

soi

l, g/

kg

landfarmingbioremPhytorem onlyMPPS

MPPS: Greenhouse 120 days

Field 120 days

Both at 50 to 60 % Remediation in 120 d

Barley/Rye 30 days - PGPR + PGPR

Sarnia Land Farm – 2005

Barley/Rye 40 days - PGPR + PGPR

Barley/Rye + PGPR - PGPR

Fall rye 30 d

- PGPR + PGPR

15 % TPH

Remediation of Metals

PGPR Effect on Plant Growth with 2% Salt in Irrigation Water

Control w/ PGPR Salt w/ PGPR Salt w/o PGPR

Plants alone (- PGPR)MPPS (+ PGPR)

Dundas foundry soil – PAH contamination ~ 500 ppm

MPPS: germinated earlier and grew faster in the contaminated soil

Plants alone (- PGPR) MPPS (+ PGPR)

Dundas Foundry: 30 d growth, MPPS has 300% more plant biomass in roots and shoots than plants alone

Effectiveness of the MPPS for Remediation From Dundas Foundry Soil: 90 days

PAH

rem

aini

ng, %

unt

reat

ed

0

20

40

60

80

100

120

Biorem Phytorem MPPS Control soil

Multi-Process System for Remediation of PAH ContaminatedBrownfield: 90 d remediation leads to ~ 40 % removal of organics

Untreated brownfield soil Bioremediation for 90 days

DBP

CHRBAP

DBA

BBA BKF

BBFPYR

FLA

PHE

BGP

Phytoremediation for 90 days MPPS for 90 days

DDT Remediation with Millet Summer 2005, Simcoe, ON

+ PGPR+ Peat Moss

w/PGPR

+ PGPR- Peat Moss

70 d growth

Remediation of DDT

• Approximately a 0.5 Hectare site• SW Ontario farmland• Homogeneous levels of DDT at ~ 0.8

mg/kg (ppm)• Half planted with Millet and half planted

with fall rye• 40 % overall DDT remediation after 90 d• DDT breakdown products (DDE or DDD)

not found • Chlorinated compounds can be degraded

by phytoremediation

0

500

1000

1500

2000

2500

3000

0 6 12 17 23Time, days

Bio

mas

s, g

Effect of PGPR on Plant Growth duringFlooding or Drought Stress

FloodingFlooding + PGPRDroughtDrought + PGPR

EP3 (a salt tol. PGPR) -PGPR UW4+EP3 (2 PGPRs)

Growth of rye grass in salt contaminated soil

Salt impacted clay-loam soil Fenn-Big Valley. Preliminary greenhouse experiment.Cl: 1200-2000 mg/ml, SAR: 11-15, ECe: 9-15 dS/m

Survival 50 % higher with PGPR

Growth of fall rye in salt contaminated soil from Fenn-Big Valley

0

10

20

Unc

onta

m.

Con

trol

soi

lN

o PG

PRU

W4

MG

3EP

3U

W4

+ EP

3U

W4

+ M

G3

Leng

th, c

m

Fall rye shoots

0

3

6

No

PGPR

UW

4M

G3

EP3

UW

4 +

EP3

UW

4 +

MG

3

Fall rye roots

50 % more survival + 20 - 30 % more growth ≅100% more biomass

MG3/EP3: PGPR from salted soils

Triticale/Barley MixYear 1: Summer 2005, North East, AB

~ 1 % TPH on a 7 Hectare site

- PGPR +PGPR70 d growth on good soil

Remediation of the NE Alberta Site• A very large site: > 7 hectares• Overall ~1 % TPH, with high variability in TPH levels (0.3 to 3

% TPH)• 6 Hectares planted: Large scale pilot remediation; All plants

(barley/fall rye mix, 2000 kg of seeds) were PGPR treated• 1 Hectare was used as a random block test for PGPR

efficacy. Barley, fall rye and triticale were used• Pilot area: 15% to 30 % remediation was observed in 70 d• Random block test (70 d), % remediation:

+ PGPR - PGPRF3 2.8 ± 0.5 - 4.2 ± 0.7F4 8.9 ± 1.3 1.9 ± 0.3Total 21.9 ± 4.7 16.7 ± 2.3F4G 32.6 ± 10.9 28.7 ± 5.3

• Consistent rates of remediation observed • Evidence of PGPR improvement, PGPR impact on par with

Turner Valley• 2006 season: Planted only + PGPR Barly/Rye on June 20.

Multi-Process Phytoremediation System (MPPS)

1. Complicated mixtures of contaminants are present in the environment

2. Many techniques based on an individual process failed or were ineffective

3. Contaminants are too toxic to plants and bacteria for remediation

4. Use and understanding of different remediation mechanisms

5. Multiple remediation kinetics resulting in effective and efficient remediation

Field Test at Talisman Energy Biopile (1 % TPH)Turner Valley Alberta

Summer 2005 – Rye/Fescue Plant Growth

- PGPR + PGPR - PGPR + PGPR

Average root depth: 21 cm

Field Test at Imperial Oil Land Farm, Sarnia ON15 % Total Petroleum Hydrocarbon (TPH)Year 1: Summer 2004 - 60 d after planting

Rye Grass - PGPR

Rye Grass + PGPR

Field Test at Imperial Oil Land Farm, 120 d

Rye Grass - PGPR

Rye Grass + PGPR

Rye Grass + PGPR

Rye Grass - PGPR