Manipulating soil and root microbiomes to

enhance bioremediation and reclamation of hydrocarbon impacted soil ecosystems

Plant-microbe associations

can assist reclamation efforts

Jim Germida

Department of Soil Science

5thANNUAL SOUTH ASIA

BIOSAFETY CONFERENCE

September 11-13, 2017

Taj West End, Bangalore, India

Overview

▪ Soil and plant microbiomes

▪ Phytotechnologies

▪ Challenges at contaminated sites

▪ Research stories

a) Abandoned Oil Well Flare Pit

b) Bitumount Provincial Historical

c) Oil Sands Mine Site

Take home message: soil and root microbiomes

can assist remediation and reclamation efforts

The Plant Root System

- allows the plant to explore the soil

- is ‘home’ to larger, more

diverse microbial populations

than are present in the bulk soil

- releases exudates that

affect microbial activity

and numbers

de Freitas and Germida. 1990. A Root tissue culture system to study winter wheat

rhizobacteria interactions. Applied Microbiology and Biotechnology, 33, 589-595

Plant growth promotion• Pathogen control• Pollutant degradation• Phytotoxicity reduction• Enhanced nutrients availability

Root Microbiome

Gaiero, McCall, Thompson, Day, Best, and Dunfield. 2013. Inside the root microbiome: Bacterial root endophytes and plant growth promotion. American Journal of Botany, 100, 1738–1750

How can we manipulate soil and plant microbiomes?

Dunfield and Germida, 2003. Seasonal changes in the

rhizosphere microbial communities associated with field grown

genetically modified canola (Brassica napus). Applied and

Environmental Microbiology, 69, 7310-7318.

2

3

Use microbial inoculants

Grow different crops, plants or rotations

Amendments to soils

Greer, Onwuchekwa, Zwiazek,

Quoreshi, Roy Salifu, and Khasa.

2011. Enhanced revegetation and

reclamation of oil sands disturbed

sites using actinorhizal and

mycorrhizal biotechnology. Pp19-

26. Mine Closure 2011 — A.B.

Fourie, M. Tibbett and A. Beersing

(eds)

Phytotechnologies, such as phytoremediation

▪ Regions of activity

• Plant tissue

• phytovolatilization

• phytodegradation

• phytoextraction

• Root zone

• phytostabilization

• rhizodegradation

• rhizofiltration

Phytovolatilization

Phytodegradation

Rhizofiltration

Phytoextraction

Rhizodegradation

Phytostabilization

TCE, MTBE, some metals

Metals, radionuclides

TCE, RDX, pesticides

Cd, Cr, Cu, Pb, Zn

PHCs, PAHs, pesticides

Metals, radionuclides

Enhanced rhizodegradation

Unplanted bulk soil

Rhizosphere soil

Type of site: well blowout (30 ha) Contaminant: crude oil (200,000 barrels)

Age of site: 51 years Remediation effort: natural attenuation

Natural attenuation

Research Strategy

▪ Characterize soil and root microbiomes at

contaminated and petroleum impacted sites.

▪ Isolate and characterize bacterial endophytes of

vegetation at these contaminated sites with a goal

of using them as bio-inoculants to assist

reclamation efforts.

New challenges for large scale technologies

Reclamation of oil sand mining sites

Alberta’s oil sands contain 1.7

– 2.5 trillion barrels of oil

Athabasca oil sands:

Oil Sands Operations, Fort McMurray, Alberta

Overburden

Oil sands

Boreal forest

3 Key Mine Closure Challenges▪ Large areas / volumes

a) 1 mine ~Imagine a box!

• Area = widest extents Saskatoon

• Depths reaching 100 m

b) Move a cubic football field/24h

▪ Challenging waste materials

a) Saline/sodic overburden, Coke, Sulphur, Sand tailings, Fine tailings, Oil affected coarse overburden

▪ High closure expectations

a) Wetlands

• fens, marshes, bogs, swamps, shallow/open water

b) Productive uplands

6 km

▪ Unprecedented scales of

land disturbance.

a) Disturbed footprint

844 km2 in 2012

b) Only 12.5% of known extractable surface mining

reserves exhausted

• Time to deplete known surface mined reserves

(2008 rates)

• > 130 years to deplete

• Final disturbed area ~ 4,800 km2 http://www.oilsands.alberta.ca/

• 7 cities the size of Bangalore (709 km2)

Oil Sands Mining - Disturbance

Research Stories

Bitumount Provincial

Historic Site

Reclaimed Tailings Pond

Abandoned Flare Pit

Abandoned Oil Well Flare Pit

Using soil amendments, plants and

associated microorgansims to

remediate contaminated field sites

Phytoremediation Sites

Site L (Carlyle, SK)

Mixed grassland/parkland

Dark Brown to Black Chernozem

Clay

Buried flare pit soil (ca. 1700 m3)

ca. 5,550 mg TPH kg-1

pH 7.8 0.3

EC = 5.9 4.8 mS cm-1

SAR = 25 12

Site M (Bruderheim, AB)

Boreal fringe

Black Chernozem to Gray

Luvisol

Sandy clay loam

Tank farm soil (ca. 1200 m3)

ca. 3,050 mg TPH kg-1

pH 8.0 0.2

EC = 1.1 0.5 mS cm-1

SAR = 6 1

The Carlyle Site

Site L (Carlyle, SK)

a) plots established in 2002

b) raised bed system

c) RCBD with 4Trt & 4 rep

d) soil amendments = gypsum, compost, straw, fertilizer

e) two unplanted treatments (w/&w/o soil amendments)

f) two planted treatments (RTDF & U of SK)

0 5 10 15 20 25 300

10

20

30

40

Easting(m)

Northing(m)

TPH(mgkg-1) 0 5000 10000 15000

Trt4

Trt2

Trt3

Trt2

Trt1

Trt4

Trt1

Trt4

Trt2

Trt4

Trt3

Trt1

Trt3

Trt1 Trt3 Trt2

Spatial distribution of PHCs in the plot area

at the Carlyle phytoremediation site

Highest initial PHC concentrations associated with the unplanted treatments (Trt 2 > Trt 1)

Lowest initial PHC concentrations associated with the planted treatments (Trt 3 Trt 4)

Change in TPH concentration with time

● Mean value (averaged across treatments)

Trt 1 (unfertilized, unplanted)

Trt 2 (fertilized, unplanted)

Trt 3 (fertilized, planted with RTDF mix)

Trt 4 (fertilized, planted with U of S mix

CTPH = 5172e-0.001089t R2 = 0.941**

Carlyle micro-plot site

▪ 4 planted treatments

a) Single plants

• Alfalfa, Altai wild rye (AWR), tall

wheatgrass (TWG)

b) Mixed plants

• Alfalfa, AWR, TWG

▪ 1 unplanted control

▪ Samples collected and analyzed at 6 week

intervals over two growing season

Site set-up

July 2006 September 2006

July 2004 September 2004

Plant effect on PHC degradation(Carlyle site, 2005 micro-plot data)

▪ Greater degradation under single species plantings

AWR Alf > TWg UnP > Mix

Phillips, Greer, Farrell and Germida. 2009. Field-scale assessment of weathered

hydrocarbon degradation by mixed and single plant treatments. Applied Soil Ecology

42: 9–17

Altai wild rye

Opportunities for grasses and legumes

Endophytic n-hexadecane degraders

Phillips, Germida, Farrell and Greer. 2008. Hydrocarbon degradation

potential and activity of endophytic bacteria associated with prairie plants Soil

Biology & Biochemistry 40: 3054–3064

Endophytes of grasses and legumes

▪ Microbial MPN analysis reveals that all mature plants maintain large populations of aliphatic and total hydrocarbon degraders in their root tissue

– PAH degraders present in

mature plant tissue at low

levels or as discrete sub-

populations

Phillips, Greer, Farrell and Germida. 2012. Plant root

exudates impact the hydrocarbon degradation potential of

a weathered-hydrocarbon contaminated soil. Applied Soil

Ecology 52: 56– 64

A survey of bacterial root endophytes associated with

vegetation at the Bitumount Provincial Historic Site

Blaine, 2016. MSc Thesis, University of Saskatchewan

Bitumount Provincial Historic Site

Bitumount

Circa: 1940s

http://www.history.alberta.ca/energyheritage/bitumount/D

efault.aspx

Plant colonization in Bitumount

Blaine, Helgason and Germida. 2017. Endophytic root bacteria associated with the

natural vegetation growing at the hydrocarbon-contaminated Bitumount Provincial

Historic site. Canadian Journal of Microbiology, 63: 502–515.

A

B

C

D

E

F

Sampling locations

Processing Area

Pathway

Riverbank

Entrance

Quarry Border

Quarry

Location pH Nitrate Sulfate Phosphate Potassium

Total

hydrocarbons

--------------------------------------mg kg-1-----------------------------------

Quarry 7.0 <1 22.1 <2 <20 24700

Entrance 8.4 2.2 12.3 5.5 36 4120

Processing Area 8.2 1.5 13.4 7.3 109 3500

Pathway 8.5 <1 4.9 3.7 31 2350

Quarry Border 8.5 <1 28.2 <2 58 1770

River Bank 8.5 <1 47.1 <2 90 330

Hydrocarbon levels according to the methods of the Canadian Council of Ministers and the

Environment (CCME)

Soil properties analysis

Vegetation sampled

Wild Strawberry(Fragaria virginiana)

Horsetail(Equisetum spp.)

Pea family(Fabaceae spp.)

Smooth Brome(Bromus inermis)

Kentucky Bluegrass(Poa pratensis)

Slender Wheatgrass(Agropyron trachycaulum)

Materials and methods

Culture independent

Sanger sequencing• 16S rRNA

Isolation on 1/10th

strength TSA mediaCulture Collection

Illumina MiSeq• 16S rRNA• 520F/799R

DNA extraction

Culture dependent

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Pea family

Slender Wheatgrass

Smooth Brome

Horsetail

Slender Wheatgrass

Smooth Brome

Kentucky Bluegrass

Smooth Brome

Wild Strawberry

Horsetail

Slender Wheatgrass

Smooth Brome

Qu

arry

Entr

ance

Pro

cess

ing

Are

aP

ath

way

Qu

arry

Bo

rder

Riv

er B

ank

Relative Abundance (%)

Rhizobiales

Burkholderiales

Pseudomonadales

Sphingomonadales

Xanthomonadales

Caulobacterales

Unclassified Gammaproteobacteria

Unclassified Proteobacteria

Unclassified Betaproteobacteria

Rhodospirillales

Unclassified Alphaproteobacteria

Enterobacteriales

Myxococcales

Desulfuromonadales

Methylophilales

Rhodobacterales

Desulfobacterales

Legionellales

Campylobacterales

Acholeplasmatales

Solirubrobacterales

Actinomycetales

Acidobacteria Gp1 (Incertae sedis)

Bacillales

Clostridiales

Sphingobacteriales

Flavobacteriales

Unclassified Bacteroidetes

Bacteroidetes (Incertae sedis)

Herpetosiphonales

<1%

Culture independent techniquesOrder

(24

70

0)(4

12

0)

(35

00

)(2

35

0)

(17

70

)(3

30

)

Horsetail Slender Wheatgrass Smooth Brome Wild Strawberry Kentucky Bluegrass Pea Family

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Quarry Entrance ProcessingArea

Pathway QuarryBorder

River Bank

Log

CFU

g-1

Fres

h R

oo

t

Rhizosphere

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Quarry Entrance ProcessingArea

Pathway QuarryBorder

River BankLo

g C

FU g

-1Fr

esh

Ro

ot

Endophytes

Enumeration of culturable bacteria

Lessons Learned

• Bitumount represents a unique opportunity for research

• Despite high hydrocarbon levels, plants were able to support a variety of root associated microorganisms

• Many of the microorganisms identified are associated with plant growth promotion and may have the potential to use as bio-inoculants to aid in reclamation efforts

Oil Sands Reclamation Strategies

2007

200820102012 2010

Mitter, de Freitas and Germida. 2017. Bacterial root microbiome of plants

growing in oil sands reclamation covers. Frontiers in Microbiology, 8:849

Sampling transects along the engineered and standard

cover at an oil sands mine site reclamation area

Cover pH TextureNH4-N NO3-N S P K

Total

Hydrocarbons*- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ( p p m ) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Engineered 7.6 Sandy loam 3.8 26.0 40.3 5.5 74.0 1390

Standard 7.4 Sandy loam 4.0 20.4 47.9 4.3 76.0 834

Physical and chemical properties of peat mineral mix collected in the

engineered and standard cover at an oil sands mine site reclamation area.

* Petroleum hydrocarbons (C5-C60) fractions according to the methods of the Canadian Council of Ministers of the

Environment (CCME).

Denaturing Gel Gradient

Electrophoresis (DGGE)

PCR

Rhizospheric

Bacteria

Surface

Disinfection

Isolation in 1/10th

TSA medium

Isolation in 1/10th

TSA medium

Phospholipid Fatty Acid

(PLFA) Analysis

Endophytic Bacteria

Culture Collection

Soil

Root samples

DNA

extraction

Pyrosequencing

Standard Cover Engineered Cover ---------- Location ------------------ Location --------

S1 S2 S3 S4 S6

< 2< 3< 4

1> > > > >

E1 E2 E3 E4 E6

<5

<6

> > > > >

Band Accession # Closest match Similarity (%)

1 EF664750.1 Uncultured proteobacterium clone GASP-MB1W1_C05 16S ribosomal RNA gene, partial sequence 99

2 FJ448589.1 Uncultured bacterium clone D1_KR_030507_G03_23_13 16S ribosomal RNA gene, partial sequence 100

3 EU593726.1 Lentzea violacea strain 173540 16S ribosomal RNA gene, partial sequence 100

4 KJ425224.1 Actinoplanes auranticolor strain INA01094 16S ribosomal RNA gene, partial sequence 99

5 EF664750.1Leafhopper (Deltocephalinae) aster yellows phytoplasma partial 16S rRNA gene, isolate Colombia

isolate L13100

6 U96616.1 Phytoplasma sp. STRAWB2 16S ribosomal RNA gene, partial sequence 98

Endophytes

0

20

40

60

80

100

Rhizosphere Endosphere Rhizosphere Endosphere

Barley Clover

Rel

ativ

e ab

un

dan

ce (

%)

Proteobacteria Actinobacteria BacteroidetesTenericutes Firmicutes GemmatimonadetesAcidobacteria Other

Analysis of root associated bacterial communities (endosphere and the rhizosphere

compartments) at phylum level in barley and sweet clover growing in oil sands

reclamation areas.

BECEBRCR

PC2(12.38%)

PC1(9.55%)

PC3(27.17%)

Principal Coordinate Analysis (PCoA) based on Bray-Curtis dissimilarity between

samples for barley endosphere (BE), clover endosphere (CE), barley rhizosphere

(BR) and clover rhizosphere (CR).

Relative abundance (%)

Vertical columns represent samples; horizontal rows represent genera that are 15 % most

abundant in at least one sample. Clustering of samples (top) is based on genera co-occurrence

by Bray-Curtis dissimilarity. Letters (A-F) indicate different clusters at a 70% dissimilarity cut off.

Bacterial endophytes as bio-inoculants to

enhance bioremediation▪ A total of 316 endophytic bacteria isolated from oil sands mine site were tested

for the presence of hydrocarbon degrading genes ( alkB, CYP153 and NAH).

▪ 42 isolates were tested positive for at least one hydrocarbon degrading gene.

Seed germination

Study 1

Root elongation

Study 2

Growth chamber

Study 3

Mitter, PhD Thesis, University of Saskatchewan

Isolate

#

Bacterial Species Gene

3 Stenotrophomonas spp. CYP 153

9 Flavobacterium spp. alKB

26 Pantoea spp. alKB

33 Pseudomonas spp. alKB

Endophytes enhance plant growth

• Sweet clover (Melilotus albus)

• 1.5 Kg of Dark Brown Chernozem silty

clay agricultural soil.

• Diesel concentrations of 5,000, 10,000

and 20,000 mg/kg of soil.

• Plants were harvest after 65 days.

Control Stenotrophomonas

spp.Flavobacterium spp. Pantoea spp. Pseudomonas spp.

Pure Diesel (50 ppm)

F2

C10 – C16

F3

C16-C34

Hydrocarbon Fractions

Control

EA1-17EA2-30

EA4-40EA6-5

5,000 mg of diesel per Kg of soil

1 M

Contro

l

EA1-17

EA2-30EA4-40 EA6-5

10,000 mg of diesel per Kg of soil

1 M

Diesel Concentration (mg·Kg-1)

Treatment 5,000 10,000 20,000

Control (unvegetated) 430.69a 5086.69a 12974.22a

Control (vegetated) 418.70a 4882.57a 12952.72a

EA1-17 429.78a 1849.09b 10976.27ab

EA2-30 468.93a 1877.28b 10261.35ab

EA4-40 431.32a 2010.31b 7625.73bc

EA6-5 426.57a 2304.31b 4163.92c

Hydrocarbon Degradation

Total soil hydrocarbon fractions (F2, F3) after bacterial inoculation on white

sweet clover plants growing in soils initially amended with diesel at 5,000,

10,000 and 20,000 mg·Kg-1 at 65 days after planting. Different letters indicate

significant differences (Tukey HSD p≤0.05).

a a

aa a

a

AA

B B BB

0

1000

2000

3000

4000

5000

6000

7000

Control

(unvegetated)

Control

(vegetated)

EA1-17 EA2-30 EA4-40 EA6-5

HydrocarbonFractions(m

g.kg-1ofSoil)

Hydrocarbon Degradation

Soil F2 and F3 hydrocarbon fractions after bacteria inoculation on white sweet clover plants

growing in soils initially amended with diesel at 10,000 mg·Kg-1 at 65 days after planting. Error

bars represent standard deviations (n=5). Different letters indicate significant differences

(Tukey HSD p≤0.05).

F2 F3 fractions

Lessons Learned

• Plants support a wide variety of root endophytic bacteria

• Abundance and community structure is influenced by the

interaction of plant species and environment

• Many bacterial endophytes enhance plant growth and

bioremediation of contaminants

Phytoremediation Research Group

U of S Faculty

Jim Germida

Rich Farrell

Diane Knight

Ken Van Rees

Bing Si

Renato de Freitas

Graduate Students

Eduardo Mitter

Natalie Blain

Jen Fernet

Adam Gillespie

Lori Phillips

Diana Bizecki Robson

Julie Roy

Shannon Gerrard

Carol Luca

Alexis McFerson

Cory Sonntag

Monique Wismer

\

Bobbi Helgason (AAFC)

John Headley (NWRI)

John Lawrence (NWRI)

Charles Greer (NRC-BRI)

Sarah Armstrong (Shell)

Funding Acknowledgements

Natural Sciences and Engineering Research Council (NSERC) –Strategic Projects Grant Program

Saskatchewan Agriculture & Food: Strategic Research Program – Soils & Environment

Environment Canada

Canadian Natural

TOTAL

Environmental Research Advisory Council

Talisman Energy

Husky Energy, Inc.

Federated Co-Operatives, Ltd.

Shell Canada

SUNCOR

Syncrude

COSIA –Canada’s Oil Sands Innovation Alliance

Program of Energy Research & Development

Canadian Association of Petroleum Producers

Millar Western