manipulating soil and root microbiomes to enhance...
TRANSCRIPT
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
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:
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
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
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.
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.
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