plants and their associated bacteria: partners in...

Plants and their associated bacteria: partners in remediation of contaminated

soils and groundwater?

Jaco Vangronsveld, Sofie Thijs, Panagiotis Gkorezis, Nele Weyens

General considerations and examples from the field

Plant-associated bacteria:

Bacteria: you can find them everywhere!

?

General feeling about bacteria…


At least half a kilogram

of our body weight are

bacteria!!!

Bacterial cells are much smaller than human cells.

There are at least 10 times as many bacteria as

human cells in our body!!

Bacteria:

the fingerprint of the future?

Bacteria: humans probiotics!

Salad for lunch?!


A plant is much more than you can see!Plant-

geassocieerde

bacteriën in

bodemsanering,

van labo tot

veldschaal

Plant-geassocie

erde bacteriën

in bodemsanering,

van labo tot

veldschaal

Plant-geassocie

erde bacteriën

in bodemsanering,

van labo tot

veldschaal

Marlene Cameron and Sheng-Yang HeMichigan State University

Bonfante and Anca Ann Rev Microbiol. 2009 63: 363-83

A plant is much more than you can see!Plant-

geassocieerde

bacteriën in

bodemsanering,

van labo tot

veldschaal

Plant-geassocie

erde bacteriën

in bodemsanering,

van labo tot

veldschaal

Plant-geassocie

erde bacteriën

in bodemsanering,

van labo tot

veldschaal

Hardoim et al., 2008

• Rhizosphere bacteria

• Endophytic bacteria

• Seed derived

• Soil derived

Bacteria: plants probiotics!

0.00

1.00

2.00

3.00

4.00

5.00

Controle W619 W619 + gfp

Root

mass

(g)

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Controle W619 W619 + gfp L

eaf

mass

(g)

Bacteria can promote plant growth

Bacteria: plants probiotics!

Indirect growth promotion: competition between

plant growth promoting bacteria

and pathogens

N2

NH4+

Direct growth promotion:

- Increase the availability of:

N: N-fixation (e.g. Rhizobium)

P: P-solubilization

Fe: Fe-solubilization

Biofertilizers

- Production of plant growth hormones

Auxins, gibberelins, cytokinins

- Production of stress reducing enzymes

e.g. ACC-deaminase

Competition between PGP-bacteria and pathogens

inoculated

non-inoculated

Remediation - phytoremediation

Soil and groundwatercontamination

Using plants and bacteriafor remediation purposes

© Max Planc institute

© Hasselt University

© Biozentrum

Universitat

Wurzburg

Phyllosphere microbiome Endosphere

microbiome

Rhizosphere

microbiome

Why soil remediation?

Soil is one of the largest reservoirs of biodiversity

Soil biodiversity

• Microorganisms (mikros organismos): small microscopic living organisms

• The majority of the bacteria are yet unknown: < 1 % of the microbial species on Earth

• 1 gram soil: contains 1,000,000,000 microbial cells and potentially 1000’s of species

• Global Biodiversity assessment: 1,000,000 bacterial species on Earth; up to now, less than 4,500 have been characterized.

What is “PHYTOREMEDIATION”?

“Using plants and their associated microorganisms to degrade, or alternatively detoxify organic and inorganic pollutants in soil, (ground)water or sediments

”

!!! Plants are mostly not the ‘exclusiveplayers in the game’. A very important rolecan be attributed to plant-associated bacteriaand mycorrhiza.

“Pump and treat”using solar energy

What is “PHYTOREMEDIATION”?

Trees can ‘pump’ big volumes of (undeep)groundwater (f.i. poplar: mean of 260 litersper tree each day; this results in anevaporation of 4.2 million liters per hectare in6 months!!!)

Phytoremediation of trace elements

Phytoextraction

Extracting metals from soil in harvestable plant biomass

Trend towards:- sustainable land-use- biofuel production

Trace elements(Cd, Ni, Cu, Zn…)

Phytostabilisation

• Stabilizing trace elements in soil:

• Reduction or even elimination of lateral wind and water erosion

• Biological ‘immobilization’ of trace elements

• Reduction of percolation

Risk-reduction

Phytoremediation of organic contaminantsPlant-

geassocieerde

bacteriën in

bodemsanering, van labo tot

veldschaal

Plant-geassocieer

de bacteriën

in bodemsane

ring, van labo tot

veldschaal

Plant-geassocieer

de bacteriën

in bodemsane

ring, van labo tot

veldschaal

Can the plant reach the contaminants?

Can the contaminants reach the plant?

Degradation

Rhizosphere

Xylem vessels

Evapotranspiration?

Phytoremediation of organic contaminantsPlant-

geassocieerde

bacteriën in

bodemsanering, van labo tot

veldschaal

Plant-geassocieer

de bacteriën

in bodemsane

ring, van labo tot

veldschaal

Plant-geassocieer

de bacteriën

in bodemsane

ring, van labo tot

veldschaal



Degradation

Rhizosphere

Xylem vessels

Evapotranspiration?

Phytotoxicity?

Bacteria: important players during phytoremediationof organic contaminants



Degradation

Rhizosphere

Xylem vessels

Evapotranspiration?

Phytotoxicity?

How can bacteria assist their hostplant?

• Growth promotion

• Production of biosurfactants,

organic acids, siderophores

• Degradation of the contaminant

- Rhizosphere:

high diversity vs short contact time

- Xylem vessels:

lower diversity vs long contact time

Constraints for phytoremediation of organics

• Can the plants tolerate the contaminants?

• Do the naturally present bacteria possess

the appropriate characteristics?

• Is the amount of interesting strains high

enough to avoid phytotoxicity and

evapotranspiration?

Phytoremediation can be applied!

Field case 1: BTEX contamination

Groundwater plume contaminated with

BTEX due to leakage of former

underground storage tanks.

1999:

- sources of contamination were eliminated

- bioscreen consisting of 275 poplar trees was

planted on the BTEX groundwaterplume

Why poplar???

1. Fast growth

2. High biomass production

3. They are phreatophytic

4. Poplar trees can “pump”

huge volumes of groundwater

5. They are simple for propagation


2000 2003 2006


Are the appropriate bacteria present and are they doing their job?

fze 2003 2006

Outside the BTEX plume

Endophytes 18 x 105 0

Rhizosphere 18 x 105 0

Inside the BTEX plume

Endophytes 54 x 105 0

Rhizosphere 10 x 106 0

Bacteria were isolated from

the roots and rhizosphere of

the planted poplar trees

and tested for their BTEX

degradation capacity

Field case 2:diesel oil contamination

poplar and willow

Leakage of diesel oil storage tank

Oil contamination plume

Poplar and willowwere planted in 2006

Field case 2:oil contamination

2004 2008

Are the appropriate bacteria present and are they doing their job?

Most

probable

number

method

Degradation capacity? Growth promotion capacity?

Production of organic acids

HPLC

Production of siderophores

Evapotranspiration?

Selected strains

Strain A

C

C

I

A

A

Organic

acids

Inorganic /

Organic P

Sid Quorum sensing

/ BS

Solvent

tolerance

Chemotaxis/

Biofilm

Antibiotics

sensitivity

Heavy

Metal

Acinetobacter

oleovorans

( Root )

+ + + + + + + + Ampicillin

Bacitracin

Pb,Cu,Zn

& Ni up to

1.5 mM

Acinetobacter

calcoaceticus

( Rhizosphere)

+ - + + + + + + Ampicillin

Bacitracin

Cl -

amphenicol

Pb,Cu,Zn

& Ni up to

1.5 mM

Brevibacterium

sp.(Rhizosphere)+ - - + - + + + Ampicillin

Bacitracin

Cl -

amphenicol

Pb, Cu ,

Zn up to

1.5 mM

and Zn up

to 4 mM

0

20

40

60

80

100

120

Initial nC10 nC11 nC12 nC13 nC14 nC15 nC16 nC17 nC18 nC19 nC20 nC21 nC22 PristanePhytane

Acinetobacter calcoaceticusSingle hydrocarbons - Residual concentrations after 10 days

Degradation data GC-MS

Deciphering the microbial “ black box”

The microbial community is responsible forcontaminant degradation.

The use of new genomics tools can help decipherthe who, what, where and how

Knowing who’s there provides importantinformation on capabilities

Knowing which key genes are present identifiespotential

Showing which genes are functional indicateswho is active

Bacterial DNA

Reads

(200bp)

Contigs

(100 to

30000 bp)

Sequencing

Genome assembly

Ion Torrent Sequencing

Genome characteristics

Size : 5.2 MBp

Acinetobacter oleivorans: 4.15 MBp

Genes: 11560

Acinetobacter oleivorans: 3963

DNA

Diesel degradation

FATTY ACID DEGRADATION

Alkane-1-monooxygenase

PAH DEGRADATIONNaphthalene dioxygenase

(Ferredoxin dependant system)

Sequenced bacteria

3 genes

3 genes

Octane 1-Octanol

Naphthalene 1,2-dihydronaphthalene

DNA

Plant growth promotion

LOWERING OF PLANT ETHYLENE PRODUCTIONACC deaminase

BACTERIAL SYNTHESIS OF IAANitrile hydratase

(IAA Biosynthesis type IV)

Sequenced bacteria

2 genes

2 genes

Indole-3-acetamideIndole-3-acetonitrile

1-aminocyclopropane-1-carboxylate 2-oxobutanoate

Quorum sensing and biofilm formation

Sequenced bacteria

Prolyl isomerase (2 genes)

N-Ac Glucosamine synthase (5 genes)

Biofilm dispersion protein (3 genes)

Penicillin amidase (1 gene)

Ornithine utilization protein (3 genes)

Acyl-homoserine lactone acylase (6 genes)

Quorum sensing Biofilm formation

DNA

Iron uptake

Sequenced bacteria

SIDEROPHORE BIOSYNTHESIS(4 genes)

•Aerobactin•Rhizobactin•Other putative siderophore

SIDEROPHORE RECEPTORS(16 genes)

•Aerobactin receptor•Ferrioxamin receptor•Ferrichrome receptor•Entrobactin receptor•Colcicin receptor•Vibriobactin receptor•Pseudobactin receptor

PROTEIN BREAKING THE Fe-SIDEROPHORE COMPLEX

(1 gene)NADPH-dependent ferric-chelate reductase

DNA


1. Can the plants tolerate the contaminants?

2. Do the naturally present bacteria have the

appropriate characteristics?

1. Is the amount of interesting strains high

enough to avoid phytotoxicity and

evapotranspiration?

Can these bacteria or their

appropriate characteristics be

enriched/introduced?


Can the appropriate bacteria be ‘constructed’

and introduced?

What would be the’ ideal’ bacterial strain for remediation of volatile organics?

Endophyte Long contact time between

contaminant and degrader

Easier to introduce

PGPB

Degrader

Degradation genes on

mobile DNA: plasmids

How to make and introduce the ideal bacterial strain?

Soil bacteria with

degradation plasmidPGP endophytes

What do we have?

The ‘ideal’ strain can be made by natural conjugation

Introduction by means of inoculation

Constraints for phytoremediation

Toluene-exposed lupine plants

are inoculated with

a toluene degrading endophyte

Phytotoxicity decreases

Evapotranspiration decreases

Can the appropriate bacteria be made and introduced?

Proof of concept:

Field case 3: TCE contamination

Mixed wood of english oak and common ash

TCE-contamination

Planted poplar trees

TC

E μ

g/l

Site background

All cultivable oak- and ash-associated bacteria were isolated,

identified and tested for TCE tolerance and degradation


82%

3%

TCE tolerance and degradation

Is this TCE degradation enough to prevent TCE evapotranspiration?

Ash: 10.84*10-3± 1.17*10-3 ngTCE/cm²h

Oak: 6.35*10-3± 0.18*10-3 ngTCE/cm²h


Poplar trees were planted on

the TCE contamination plume

The trees were equipped with a drainage tube for inoculation

Which bacterial strain was inoculated?

The TCE degrading,

plant growth promoting

poplar endophyte

Pseudomonas putida W619

TCE genes

Mixed wood of english oak and common ash

TCE-contamination

Planted poplar trees

TC

E μ

g/l


0

1

2

3

4

5

6

7

8

9

10

Control W619

10

-2 n

g c

m-2

h-1

0

1

2

3

4

5

6

7

8

9

10

Control W619

10

-2 n

g c

m-2

h-1

Before inoculation 3 months after inoculation

The in situ TCE evapotranspiration was measured

Total number of cfu per g fresh weight:

9.4 x 104 3.2 x 107

Relative abundance (%)

ddddddddd

dBacillus

ddddddddd

d

Rhizobium

Dddddd

dddd

Pseudomonas

Blanc

Pseudomonas putida W619-TCE

Pseudomonas spp. from inoculated roots

Pseudomonas spp. from non-inoculated roots

1kb DNA-ladder

Non-inoculated trees Inoculated trees

Establishment and enrichment of P. putida W619

0

5

10

15

20

25

30

35

Pseudomonas putidaW619-TCE

Pseudomonas spp. frominoculated roots

Pseudomonas spp. fromnon-inoculated roots

TCE

de

grad

atio

n(%

)

ROOT

Weyens et al., 2009

Field case 3: TCE contaminationWere the inoculated strain and its degradation genes introduced successfully?

Weyens et al., 2009

Relative abundance (%)

yyyyyyyyyyyyy

yy

Curtobacterium

yyyyyyyyyyyyy

yy

Frigoribacterium

Non-inoculated trees Inoculated treesTotal number of cfu per g fresh weight:

1.0 x 103 5.4 x 104

STEM

0

5

10

15

20

25

30

35

40

Pseudomonas putida

W619-TCE

Frigoribacterium spp. from

inoculated stems

Frigoribacterium spp. from

non-inoculated stems

TC

E d

egra

dati

on

(%

) Horizontal

Gene transfer

Field case 3: TCE contaminationWere the inoculated strain and its degradation genes introduced successfully?


Were the inoculated strain and its degradation genes introduced successfully?

Horizontal gene transferTCE genes

P. putida W619

In situ inoculation

Roots:The inoculated P. putida W619 with the TCE

genes was re-isolated in very high numbers

Stem:

But

3 months after inoculation

The inoculated P. putida W619

with the TCE genes could not

be re-isolated

The inoculated TCE degradation

genes were found in other, natural

abundant stem endophytes

Conclusions

Can the tree reach the contaminants?

Can the contaminants reach the tree?

Degradation

Evapotranspiration?

Phytotoxicity?

1) Degradation capacity is present

in the natural abundant population

Degradation genes

Conclusions



Degradation

Evapotranspiration?

Phytotoxicity?

2) Degradation capacity is NOT present


INOCULATION

Degradation genes

Conclusions



Degradation

Evapotranspiration?

Phytotoxicity?

Degradation genes



INOCULATION

Enrichment of the inoculated strain

Conclusions

Transfer of the degradation genes

Degradation genes



INOCULATION



Degradation

Evapotranspiration?

Phytotoxicity?

Phytodegradation/detoxification

Plants and microbiota transform, degrade, detoxify organic pollutants:

- In planta (highly water-soluble compounds)

- Rhizosphere (rhizoremediation)

Organic pollutants

Human intervention

“Pump and treat”using solar energy

General conclusions: phytoremediation of organic pollutants

1. Selecting the phytoremediator plant type

2. Exploiting the plant microbiome

3. Improving contaminant bio-availability

Phytoremediation: very promising, cheap and sustainable clean-up method

Barac, T., Borremans, B., Provoost, A., Oeyen, L., Colpaert, J.V., Vangronsveld, J.,Taghavi, S., van der Lelie, D. (2004) Engineered endophytic bacteria improvephytoremediation of water-soluble volatile organic pollutants. Nature Biotechnology,22, 583-588.

Barac, T., Weyens, N., Oeyen, L., Taghavi, S., van der Lelie, D., Dubin, D., Spliet, M.,Vangronsveld, J. (2009) Application of poplar and its associated microorganisms forthe in situ remediation of a BTEX contaminated groundwater plume. InternationalJournal of Phytoremediation, 11, 416-42

Weyens N, Taghavi S, Barac T, van der Lelie D, Boulet J, Artois T, Carleer R,Vangronsveld J (2009) Bacteria associated with oak and Ash on a TCE-contaminatedsite: characterization of isolates with potential to avoid evapotranspiration.Environmental Science and Pollution Research, 16: 830-843

Weyens N, Boulet J, Adriaensen D, Timmermans J-P, Prinsen E, Van Oevelen S, D’HaenJ, Smeets K, van der Lelie D, Taghavi S, Vangronsveld J. (2012) Contrastingcolonization and plant growth promoting capacity between wild type and a gfp-derativeof the endophyte Pseudomonas putida W619 in hybrid poplar. Plant and Soil, 356:217–230

Weyens N, Truyens S, Dupae J, Newman L, van der Lelie D, Carleer R, Vangronsveld J.(2010) Potential of Pseudomonas putida W619-TCE to reduce TCE phytotoxicity andevapotranspiration in poplar cuttings. Environmental Pollution, 158: 2915-2919

Weyens N, van der Lelie D, Artois T, Smeets K, Taghavi S, Newman L, Carleer R,Vangronsveld J (2009) Bioaugmentation with engineered endophytic bacteria improvescontaminant fate in phytoremediation. Environmental Science and Technology, 43:9413-9418

Part of the results shown in this presentation are published in:

plants and their associated bacteria: partners in...

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