PHYTOREMEDIATION ANovelTechnologyto

DecontaminatePollutedSites

NicolasKalogerakis
DepartmentofEnvironmentalEngineering
TechnicalUniversityofCrete

Coworkers:
J.Kadukova,EManousaki,M.Kokkali,
M.Nikolopoulou

Phytoremediation
Phytoremediation isdefinedastheuseofgreenplantsandtheir
associatedmicroorganisms,soilamendments,andagronomic
techniquestoremove,degradeordetoxifyharmfulenvironmental
pollutants.

Phytoremediationtechnologies:
Phytoremediationtechnologies:
I. RhizosphereEnhancedBioremediation(orPhytostimulation)
II. Phytodegradation(orPhytotransformation)
III. Phytostabilization
IV. Phytoextraction(orPhytoaccumulation)
V. Rhizofiltration
VI. Phytovolatilization
VII. Phytoexcretion(?)
 Phytoremediation processes
Phytoextraction: transfer of
pollutants from the soil and
accumulation in the above
ground parts of the plant.
Phytovolatilization : transfer of
pollutants from the soil to the
atmosphere.
Phytodegradation:
enzymatic degradation of the
pollutants in the plant tissue.

Rhizofiltration: transfer
of pollutants from the soil
Phytostabilization:
and accumulation in the
Stabilization of heavy
roots of the plant.
metals in the soil/root
surface and reduction of
heavy metal mobility.
Enhanced Bioremediation (or Phytostimulation ):
Enhancement of the microbial community and increase of
biodegradation in the rhizosphere.

PhytoremediationResearchatTUCrete

GeneralProject:
Phytoremediationofcontaminatedsiteswithheavymetalsusing
Mediterraneanplants.

Specificaims:
Heavymetals:Lead(Pb),Cadmium(Cd)andtheirmixtures.
IdentificationofPbandCdhyperaccumulatorsamong
Mediterraneanplants
Focusingonsalt
Focusingonsalttolerantplants
 Why halophytes??
Halophytescanbecultivatedwithsalineirrigationwater
whichisadesirablefeaturesinceoftenhighqualityirrigation
waterisnotavailableevenforapplicationtocropsinaridand
semiaridregions.
Saltwaterirrigationisbecominganincreasinglyimportant
practicebecausethequalityofirrigationwatersisdecreasing
aswatersuppliesforagriculturebecomerestricteddueto
urbanneedsandclimatechange.
Salinity hasbeenshowntobeakeyfactorfor
theincreasedbioavailabilityofmetalsinthesoilsdueto
reducedsoilmetalsorption
thetranslocationofmetalsfromrootstotheaerialparts
oftheplant animportantfeatureforphytoextraction
applications

Salttolerantplantsexamined:

Plant#1: Tamarixsmyrnensis

Plant#2: Neriumoleander

Plant#3: Atriplexhalimus
 Tamarix Experiments
Potexperiments withplantsgrowninmetalpollutedsoils in
orderto evaluatetheeffectofmetalsandsoilsalinityonthe
growthofplant

Measurements:
Plant Soil
Biomass Totalmetals
Height Plantavailable
Watercontent metals
Chlorophyll pH
Proteins EC
Peroxidaseactivity Organicmatter
Metalcontent(in TotalCaCO3
rootsandshoots)

PotExperiments ExperimentalConditions

T.smyrnensis growingincontaminatedsoilwith
800 ppmPband 16 ppm Cd

1015cmcuttingsofT.smyrnensis

Propagationperiod:21days

Adaptationperiod:8months

Experimentalperiod:10weeks

Temperature:19 47C

Humidity:18 70%

Photoperiod:1415h
 Tamarixsmyrnensis

Pb accumulationintheplant
Pb

500
450 Shoots
Pb concentration in plant tissue

400 Roots
350
300
(ppm)

250
200
150
100
50
0
0/0 0/0.5% 0/3% Pb+Cd/0 Pb+Cd/0.5% Pb+Cd/3%
Treatment

CdAccumulation
Cd concentration in plant

5,5
5 Shoots
4,5
Roots
tissue (ppm)

4
3,5
3
2,5
2
1,5
1
0,5
0
0/0 0/0.5% 0/3% Cd/0 Cd/0.5% Cd/3%
Treatment

Cd concentrationinindividualpartsofT.smyrnensis
concentrationinindividualpartsofT.smyrnensis atdifferentsoil
salinities
Salinity [%] L/R
0 0.35
0.5 0.82
3 1.4
 Tamarixsmyrnensis
25
Roots
20 Shoots
Biomass dry weight (g)

15
Biomass(dryweight)
10
Treatmentwithmixtureof Pb&
5 Cdatdifferentsalinities
0
0/0% 0/0,5% 0/3% Pb+Cd/0% Pb+Cd/0,5% Pb+Cd/3%

Treatment

Total chlorophyll Chlorophyll a Chlorophyll b

1,6
1,4
Chlorophyll content (mg/g) 1,2
1
Chlorophyll intheleaves 0,8

TreatmentwithCdatdifferent 0,6
0,4
salinities 0,2
0
0/0 0/0,5% Cd/0 Cd/0,5%
Treatment

Tamarix:Saltcrystalsonleaves
A B

Dropletssecretedbysaltglandswerecrystallizedontheleaves
duetohighTemperatures.
CdExcretionbytheLeaves
1,4

.
Amount of Cd exuded on the leaf 1,2
surface (g/g of dry leaves)
1,0

0,8

0,6

0,4

0,2

0,0
0/0 0/0.5 Cd/0 Cd/0.5
Treatment

CadmiumexcretionfromleaftissueofT.smyrnensis
CadmiumexcretionfromleaftissueofT.smyrnensis (pot
experiment).Comparisonofcontrolplantsandplanttreated
with16ppmCdofdryweightofsoilattwosoilsalinities
(0%and0.5%)

Pb&CdExcretionbytheLeaves
0,9
Metal concentration on the leaf
surface (g/g of dry leaves)

0,8 Pb
0,7 Cd
0,6
0,5
0,4
0,3
0,2
0,1
0
0/0 0.5/0 0/Pb+Cd 0.5/Pb+Cd
Treatment

Metalsexcretedontheleafsurfaceof
MetalsexcretedontheleafsurfaceofT
T. smyrnensis (pot
experiment).
experiment).Comparisonofcontrolplantsandplanttreatedwith
Comparisonofcontrolplantsandplanttreatedwith
800ppmPband16ppmCdofdryweightofsoilattwosoil
800ppmPband16ppmCdofdryweightofsoilattwosoil
salinities(0%and0.5%)
 HeavyMetalTolerance

Plantmechanisms
Plantmechanisms of heavy metaltolerance:
metaltolerance:
i. Avoidance
ii. Exclusion
iii. Immobilization
iv. Excretion
v. Mechanismsinvolvingenzymaticchanges

The resistance of halophytes to salt stress is usually

correlated with a more efficient antioxidant system (Zhu et
al.,2004).
Thus, halophytes may be more capable to cope with heavy
metals stress than common plants since heavy metal stress
inducesoxidativestresstocellularstructures.

Excretion mechanism
Salt secretion through salt glands is considered as an
adaptive strategy to regulate plant tissue ion concentration

An important mechanism which contributes to the

resistance of all plants to increased salinity levels.

Halophytes are adapted to saline environments:

salt avoidance
salt tolerance
salt evasion

The main function of salt glands is the secretion of excess

stress-
stress-inducing ions that invade the plant
 Tamarixsmyrnensis

Speciesofthegenus
SpeciesofthegenusTamarix
Tamarix arewellknownassalt
arewellknownassalttolerant plants
withtheabilitytoexcreteexcesssaltassaltdropletsthroughsalt
withtheabilitytoexcreteexcesssaltassaltdropletsthroughsalt
glandsontheirleafsurface.
glandsontheirleafsurface.

ThereisevidencethatthesaltglandsofTamarixsp.
ThereisevidencethatthesaltglandsofTamarixsp.secretewith
secretewith
minimalselectivity avarietyofdifferentions
avarietyofdifferentions andthatthe
compositionofthesecretedsaltsisrelatedtothecompositionin
compositionofthesecretedsaltsisrelatedtothecompositionin
therhizosphere.

Tamarixsmyrnensis

Transverse
sectionofthe
leaf of
T.smyrnensis
withsaltgland
 SaltcrystalsonleaftissueofT.smyrnensis at
differentsoilsalinities

0%salinity 0.5%salinity

Hydroponicexperiment ExperimentalConditions
Hydroponicgrowth withexposureto
100 ppmPband5ppm Cd
Ageofplants:
Ageofplants:10
10 months
Experimentalperiod:2
Experimentalperiod:2weeks
weeks
Temperature:
Temperature:19
19
24
24C
Humidity:
Humidity:57
57
66%
Photoperiod:
Photoperiod:12h
12h
Nutrientsolution (mg/l)
mg/l):
143.0 Ca(NO3)2 2.86 H3BO3

35.75 KNO3 1.86 MnCl2.4H2O

17.75 KCl 0.22 ZnSO4.7H2O

35.75 KH2PO4 0.079 CuSO4.5H2O

35.75 MgSO4 0.6 FeSO4.7H2O

Hydroponic experiment Measurements

Excretionratesofthemetals weremeasuredbycleaning
residuesoffleafsurfaces:
Theareabellowtheplantwascoveredbyweightedtissue
paper.Inthe3rd,6th,9th,12th and14th day theleaveswere
washedwith0.1%v/vHNO3 andtheresultingsolutionwas
absorbedbythepaper.

Metalcontentanalysisinthepaperwipes wasperformedbyICPaccording
tomodifiedmethodofSoon

Metalcontentanalysisintheplanttissue wasperformedbyICP
spectroscopyaccordingtomodifiedmethodofSoon

DeterminationofPbandCdcontentinthenutrientmedium was
performedbyICPspectroscopy

PbExcretionRates

1,6
Pb Excretion rate (g g d )
-1

1,4
-1

1,2
1
0,8
0,6
0,4
0,2
0
3 6 9 12 14
Day

Pbexcretionfromleaftissueof T. smyrnensisexposed
smyrnensisexposed to100ppm
to100ppm
Pb and5ppmCd(hydroponicexperiment)
 Phytoextraction ofcontaminatedsoilswith
heavymetals

ProblemsofPhytoextraction

Contaminatedcropdisposal
Remediationtimerequired

Phytoexcretionprocessshouldbekeptinmind

Ifnotproperlyaddressed,it reducesthe
effectivenessofotherphytoremediation
processes

Phytoextraction+Phytoexcretion

Cd Cd
Cd
Cd Cd

Opportunityto Cd Cd
Cd
intervene(?)
Cd Cd Cd Cd Surfaceaccumulation
Cd
Cd
Cd Cd
Cd Cd
Captureandremoveon
Cd Cd
Cd appropriatemedia
 Phytoremediation processes:
Phytoexcretion:
Excretion of heavy metals Phytovolatilization
from the leaves

Phytoextraction
Phytodegradation

Rhizofiltration
Phytostabilization

Enhanced Bioremediation (or Phytostimulation )

Phytoexcretion:
ANovelApproachofPhytoremediation(?)
Phyto
PhytoExcretion
Excretion:
Theplantcanbeviewedasa
Theplantcanbeviewedasabiologicalpump
biologicalpump forheavy
metals
Interveningandcapturingthedropletsonsuitablemedia
beforetheyarerecycledontothetopsoil

Advantages:
Advantages:
Thefrequencyoftreepruninganduprootingislowered
lowercosts
fasterremediationtimes
possibilityofrecoveryofmetals
Coupledtophytoextraction
 PlanningofExperimentalpart

Plant: Nerium oleander

Cd(0,0.5,3%NaCl)

Potexperiments Pb(0,0.5,3%NaCl)
(10weeks)
Pb& Cd(0,0.5,3%NaCl)

Pbincreasingconcentrations
(0,0.5,3%NaCl)

Pbincreasingconcentrations
Hydroponicexperiments
(2
(24weeks) Cdincreasingconcentrations

Neriumoleander

Pbconcentration(mgkg1 dryweight)inindividual
plantsparts

450
Pb concentration in plant

400 Shoots
Roots
350
tissue (ppm)

300
250
200
150
100
50
0
0 800 1600 2400
Pb concentration added in soil (ppm)
 Neriumoleander

.
100

Biomass dry weight (g)

90 Roots
80 Shoots
70
60
Biomass(dryweight)of 50
40
Neriumoleander 30
20
10
0
0 800 1600 2400
Pb concentration added in soil (ppm)

Total chlorophyll Chlorophyll A Chlorophyll b

Chlorophyll content (mg/g)

0,5

0,4

0,3

0,2
Chlorophyllcontents
0,1

0 ofNeriumoleander
0 800 1600 2400
Pb concentration added in soil (ppm)

Neriumoleander
Istheplantunderstress??
EffectofPbonperoxidaseactivityofNeriumoleander

1,8
Specific POD activity

1,6
(U/mg of protein)

1,4
1,2
1
0,8
0,6
0,4
0,2
0
0 800 1600 2400

Pb concentration added in soil (ppm)

 InteractionsbetweenAphids(Aphisnerii)and
OleanderGrowingonPbandCdContaminatedSoil
A

Portion of plants N. oleander infested by aphids during weeks

5 to 7 and 8 to 10 are almost identical for all treatments.
(Plants infestation recording: no presence of aphids, plant infested by number
of aphids from 1 10 and plant infested by >10 aphids)

Weeks 5 to 7 Weeks 8 to 10
 PortionofplantsN.oleander not infestedbyaphids(P(X=0))for
varioustreatmentswithleadandcadmiumasafunctionofsalinity.
Portionsmarkedwiththesameletteraresignificantlydifferent witheachother
(correspondingtodifferentsalineconcentrations)atleastat5%levelofsignificance.

Overviewofexperimentalresults
Tamarixsmyrnensis:
Suitableforphytoextractioninenvironmentswithincreased
salinity.

Neriumoleander:
Averygoodchoiceforphytostabilization.

Atriplexhalimus:
AnewPbhyperaccumulator(?)
Kadukova,J.,andN.Kalogerakis,LeadaccumulationfromnonsalineandsalineenvironmentbyTamarix
smyrnensisBunge,EuropeanJournalofSoilBiology,43,216223(2007).
Manousaki,E.,J.Kadukova,N.PapadantonakisandN.Kalogerakis,PhytoextractionandPhytoexcretionofCdby
theLeavesofTamarixSmyrnensis GrowingonContaminatedNonSalineandSalineSoils,Environmental
Research,106,326332(2008).
Kadukova,J.,E.ManousakiandN.Kalogerakis,PbandCdAccumulationandExcretionbySaltCedar(Tamarix
smyrnensis Bunge),InternationalJournalofPhytoremediation,10,3146(2008).
 Phytoremediation of organics
(OMW)
TOP VIEW VERTICAL VIEW

3 2
m m

Perforated gravel
0.50 m
water pipe

OMW >0.30 m

Subsurface
disposal area
of OMW
Protecting the river (riparian zone)
Aim: To stop the pollutant plume and
degrade contaminants that have been
extracted by the plants
Monitoring through multilevel wells

>90%
efficiency

APPLICATION: Poplars to control the flow of

nitrates from the agricultural land next to
Evrotas river.

LIFE
Environment
 Remediationofsalinesoils
Salinization isoneofthemostseriousproblemsconfronting
sustainableagricultureinirrigatedproductionlandsinsemi
aridandaridregions.UNEPestimatesthat~20%of
agriculturallandand50%ofcroplandintheworldissalt
stressed(Ravindranetal.,2007)
Soilsneedproperamendmentsasasourceofcalcium(Ca2+)
toreplacesodium(Na+)fromthecationexchangesites.The
displacedNa+ isleachedfromtherootzonethroughexcess
irrigation(Qadiretal.,2003).[Chemicalremediation
Potentialaquiferproblems?]
Canphytoremediationhelp?

Phytoremediationofsalinesoilsby
halophytes
Phytoremediationdesalinationapproach#1
Cultivationofcertainsalttolerantplantspecieswiththeabilityto
increasethedissolutionofsoilcalcite(CaCO3)intherhizosphereto
provideCa2+ thatcanbeexchangedwithNa+ atcationexchangesites.
DisplacedNa+ canbeleachedoutofthesoilwithirrigationwater.
(QadirandOster,2002;Qadiretal.,2003;Qadiretal.,2004;
Gerhardtetal.,2006)[Aquiferproblems?]

Phytoremediationdesalinationapproach#2
Halophytescouldbegrownonsaltaffectedsoilstoremovesignificant
amountsofsaltandNa+ throughtheiraerialparts.Saltisremoved
fromthesoiltotheextentthatsoilcanbereturnedtoagricultural
productivity(Chaudhrietal.,1964;GritsenkoandGritsenko,1999;
Owens,2001;KeifferandUngar,2002;Gerhardtetal.,2006;
Ravindranetal.,2007).
 CONCLUDINGREMARKS
There is a group of plants (halophytes) that have the capability to
excreteheavymetalsfromtheirleavesasadetoxificationmechanism.
excreteheavymetalsfromtheirleavesasadetoxificationmechanism.
Inthiscase,theplantbecomesa
Inthiscase,theplantbecomesabiologicalpump
biologicalpump forheavymetals.
Phyto
Phytoexcretion
excretion is an alternative phytoremediation process that
shouldbefurtherexplored.
The use of halophytes for phytoremediation applications should be
be
furtherexplored:
Rhizodegradation of organic contaminants [they can deal better
withstress]
Rhizosphereenhancedbioremediationofmixedpollutants(metals
+organics)[byremovingthemetalsthemicrobesworkbetter]
Soildesalination[alowcostlongtermremediationapproach]

Theprojectwasco
Theprojectwascofundedby
1) TheEuropeanSocialFund&NationalResources
EPEAEK IRAKLITOS)
(EPEAEK
2) MarieCurieDevelopmentHostFellowshipprogramme