Phytoremediation of PCBs and PAHs with Phytoremediation of PCBs and PAHs with Monoterpene Producing PlantsMonoterpene Producing Plants

David Crowley, David Crowley, JoongJoong--WookWook Park, Park, HaakrhoHaakrho YiYiDepartment of Environmental SciencesDepartment of Environmental Sciences

University of California, RiversideUniversity of California, Riverside

XenobioticXenobiotic

PlantPlantUptakeUptakeMetabolismMetabolismVolatilizationVolatilizationCarbonCarbon

exudateexudatequantityquantitycomponentscomponents

SurfactantsSurfactantsO2, CO2, pHO2, CO2, pHNitrogenNitrogen

Rhizosphere CommunityRhizosphere CommunitySoilSoil

SolubilitySolubilityAdsorptionAdsorption

% OM% OMClayClay

Organic matterOrganic matterTextureTexture

water contentwater contentaerationaeration

TemperatureTemperatureNutrient ContentNutrient Content

Species CompositionSpecies CompositionPopulation numbersPopulation numbers

DegradersDegradersCometabolizersCometabolizers

ActivityActivityCataboliteCatabolite repressionrepressionGene recombinationGene recombinationPlasmid transferPlasmid transfer

MineralizationMineralizationMetabolite FormationMetabolite Formation

Phytochemical Analogs of Xenobiotic Contaminants

Terpenes:Terpenes:

FlavonoidsFlavonoids

LigninLignin

QuinonesQuinones

AlkaloidsAlkaloids

PyrazinesPyrazines

NapthaleneNapthalene

FluoreneFluorene

PhenanthrenePhenanthrene PyrenePyrene

Structures of Common Aromatic Soil PollutantsStructures of Common Aromatic Soil Pollutants

Polychlorinated BiphenylsPolychlorinated Biphenyls

++

++

++

biphenylbiphenyl

PCBPCB

biphenyl +biphenyl +PCBPCB

growthgrowth

no growthno growth

growth + growth + degradation of degradation of PCBsPCBs

bacteriabacteria

bacteriabacteria

bacteriabacteria

Cometabolism of PCBsCometabolism of PCBs

OH

OH

COOHO

OH

COOH

COOH

BiphenylBiphenyl BiphenylBiphenyldioldiol

phenylhexadienoatephenylhexadienoatering fission productring fission product

hydrolysis hydrolysis productsproducts

Assay for Cometabolism of PCBsAssay for Cometabolism of PCBs

OH

Colorimetric AssayColorimetric AssayAbs. 434 nmAbs. 434 nm

Plant Screening AssayPlant Screening AssayGilbert and Crowley 1996Gilbert and Crowley 1996

Spearmint (Spearmint (MenthaMentha spicataspicata))Active component: carvoneActive component: carvone

ArthrobacterArthrobacter B1BB1B

Evidence:Evidence:PCB ring oxidation productPCB ring oxidation productchlorobenzoate product formationchlorobenzoate product formationdisappearance of disappearance of AroclorAroclor 12421242

00

10102020

30304040

5050

60607070

80809090

100100

diCBdiCB tritri--CBCB tetratetra--CBCB pentapenta--CBCB total PCBtotal PCB

PCB congener typePCB congener type

PCB

rem

aini

ng (%

)PC

B r

emai

ning

(%) Carvone induced cellsCarvone induced cells

NonNon--induced cellsinduced cellscarvonecarvone--fructosefructoseindigenous bacteriaindigenous bacteria

Degradation of 100 ppm Degradation of 100 ppm AroclorAroclor 1242 PCBs in Soil after 9 Weeks1242 PCBs in Soil after 9 WeeksAfter Inoculation With CarvoneAfter Inoculation With Carvone--Induced Induced ArthrobacterArthrobacter B1BB1B

O

O O

OHOO

O OFatty Acid Composition:

Oleic acid 74%Linoleic acid 7%Linolenic acid 2%Palmitoleic acid 7%Palmitic acid 10%

Criteria:Criteria:

NontoxicNontoxic

Selective Growth SubstrateSelective Growth Substrate

SolubilizesSolubilizes PCBPCB

BiodegradableBiodegradable

Development of a Field Application VectorDevelopment of a Field Application Vector

Sorbitan TrioleateSorbitan Trioleate

00

10102020

30304040

5050

60607070

80809090

100100

diCBdiCB tritri--CBCB tetratetra--CBCB pentapenta--CBCB total PCBtotal PCB

TreatmentTreatment

Perc

ent D

egra

datio

nPe

rcen

t Deg

rada

tion

Arthrobacter B1B cellsArthrobacter B1B cellsCarvone surfactant Carvone surfactant -- indigenousindigenouscontrolcontrol

Degradation of Degradation of AroclorAroclor 1242 PCBs after 9 Weeks of 1242 PCBs after 9 Weeks of Repeated Inoculation Using CarvoneRepeated Inoculation Using Carvone--Surfactant Grown CellsSurfactant Grown Cells

Induction of PCB degradation by Carvone and Related Compounds

PCB ring fission product formation by Arthrobacter B1B following enzyme induction by carvone or biphenyl. (Gilbert and Crowley 1998)

CarvoneBiphenyl

ngpr

oduc

t mg-

1pr

otei

n h-

13000

2000

1000

0

0 400 800 1200 1600µmol

Salicylate Salicylate cumenecumene limonene cymene carvonelimonene cymene carvone

cineole camphor cineole camphor pinenepinene vanillin vanillin

biphenylbiphenyl

citralcitral

nariniginnarinigin

O

OHO

OO

O OHOO

HO

HO

OHHO

HO

O

OO

HOO

O

OH

OHO

BRDU Labeled 16S rDNA Profile of Terpene BRDU Labeled 16S rDNA Profile of Terpene EnrichedSoilEnrichedSoil

Bip

heny

lB

iphe

nyl

Van

illi

nV

anil

lin

Citr

alC

itral

Pine

nePi

nene

Lim

onen

eL

imon

ene

Nar

ingi

nN

arin

gin

Cum

ene

Cum

ene

Cym

ene

Cym

ene

Cin

eole

Cin

eole

Car

vone

Car

vone

Cam

phor

Cam

phor

Fruc

tose

Fruc

tose

Con

trol

Con

trol

Correspondence Analysis of BRDU Labeled 16S rDNA Correspondence Analysis of BRDU Labeled 16S rDNA from Terpene Enriched Soilsfrom Terpene Enriched Soils

cumenecumene

camphorcamphor

carvonecarvonelimonenelimonene

pinenepinene

biphenylbiphenylcymenecymene

citralcitral

naringinnaringin

vanillinvanillin

fructosefructose

cineolecineole

controlcontrol

27%27%

17%17%

OO

OOOO

O O OO

OO OOOOOO

OO

OO

OOOO

OO

OO

OO

OOOO

OO

OO

OO

Salicylate: Plant Signal Compound, Siderophore,Salicylate: Plant Signal Compound, Siderophore,and Inducer of Xenobiotic Degradation Enzymesand Inducer of Xenobiotic Degradation Enzymes

Toluene dioxygenase substrates:Toluene dioxygenase substrates:

Benzene, toluene, Benzene, toluene, xylenexylene (BTEX)(BTEX)

Trichloroethylene (TCE)Trichloroethylene (TCE)

Trinitrotoluene (TNT)Trinitrotoluene (TNT)

Naphthalene, Naphthalene, benzopyrenebenzopyrene (PAH)(PAH)

Polychlorinated biphenyls (PCB)Polychlorinated biphenyls (PCB)

PCB congener PCB congener ArthrobacterArthrobacter RalstoniaRalstoniacoinoculationcoinoculation

2,3,6,2'2,3,6,2' 34 34 ±± 55 45 45 ±± 662,5,2',5'2,5,2',5' 29 29 ±± 33 56 56 ±± 772,4,2',5'2,4,2',5' 27 27 ±± 33 49 49 ±± 662,4,2',4', 2,4,5,2'2,4,2',4', 2,4,5,2' 30 30 ±± 55 44 44 ±± 662,3,2',3'2,3,2',3' 30 30 ±± 66 51 51 ±± 662,3,5,2',4', 2,4,5,2',52,3,5,2',4', 2,4,5,2',5 13 13 ±± 44 25 25 ±± 44

Coinoculation with Arthrobacter sp. B1B and Ralstonia eutrophus H850

Summary

�� Gram positiveGram positive bacteria induced by bacteria induced by monoterpenes.monoterpenes.

�� Gram negativeGram negative bacteria induced by bacteria induced by salicylic acid.salicylic acid.

�� Many indigenous PCB degraders can be enriched Many indigenous PCB degraders can be enriched by growth on these substances.by growth on these substances.

Common terpene producing plant species

Origanum majorana O. syriacumO. vulgare Salvia cabulicaThymus zygis T. piperellaCurcuma longa Carum copticumEucalyptus species Tanacetum partheniumVitex negundo Acanthopanax trifoliatusLigusticum porteri Ocimum basilicumAndropogon sp. Chenopodium sp.

Alpha-pinene, Aritasone, Ascaridole, Ascorbic-acid, Beta-carotene, Butyric-acid, Calcium, D-camphor, EO, Ferulic-acid, Geraniol, L-pinocarvone, Leucine, Limonene, Malic-acid, Menthadiene, Methyl-salicylate, Myrcene, Niacin, P-cymene, P-cymol, Phosphorus, Safrole, Saponins, Spinasterol, Tartaric-acid, Terpinene, Terpinyl-acetate, Terpinyl-salicylate, Thiamin, Triacontyl-alcohol, Trimethylamine, Urease, Vanillic-acid

Chenopodium ambrosioides

Phytoremediation of Pyrene using Celery RootPhytoremediation of Pyrene using Celery Root

Root essential oil:ppm

β pinene 15,000carvone 5000dihydro-carvone 5000P-cymene 31,000 limonene 117.000 myrcene 18,000 terpenoline 33,000trans-ocimine 290,000 cis-ocimine 68,000

Duke, J. A. 1992. Handbook of phytochemical constituents of GRAS herbs and other economic plants. Boca Raton, FL. CRC Press.

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

5 10 15 20 25 30 35 40 45

Days

ppm

Degradation of pyrene in soil amended with celery rootDegradation of pyrene in soil amended with celery root

Celery rootmg/g soil

10100200

Yardwastemg/g soil

10100200

Degradation of pyrene in the rhizosphere of wheat and celery plants

0.00.0

20.020.0

40.040.0

60.060.0

80.080.0

100.0100.0

11 22 33 44 55 66 77 88 99 1010 1111

WeekWeek

Pyre

ne (p

pm)

Pyre

ne (p

pm)

CeleryWheatControl

Selective inhibitors suggest Gram + bacteria are Selective inhibitors suggest Gram + bacteria are predominant pyrene degraders induced by celery root.predominant pyrene degraders induced by celery root.

00

2020

4040

6060

8080

100100

ControlControl CeleryCeleryrootroot

StrepStrep.. CycHexCycHex S+CS+C

Pyre

ne (p

pm)

Pyre

ne (p

pm) Gram �

Gram +Fungi

Gram �Gram +Fungi

Gram �Gram +Fungi

55--OxoOxo--1,21,2--campholidecampholide

camphorcamphor

5 5 exoexo--hydroxycamphorhydroxycamphor

camphor 5camphor 5--monooxygenase monooxygenase (cytochrome P450 cam)(cytochrome P450 cam)

2,5 2,5 diketocamphanediketocamphane

5-exo-hydroxycamphor dehydrogenase

camphor 1,2camphor 1,2--monooxygenase monooxygenase ((Baeyer-Villiger monooxygenase)

What enzymes are induced by monoterpenes?Biodegradation of Camphor: Role of P450CAM

Pairwise sequence alignment scores for α subunits of ring hydroxylating oxygenases (Nam et al 2000)

Analine

Toluene

PAH /Terpene

Biphenyl

DitANidANahAc G4PahAcDoxBNdoBNahAc 9816-4PahA3NtdAcDntAcNagAcPhnAc RP007PhnAc AFK2

96 VTLNVOPHRGMRISTADCGNTQ-IHKOIYHGWAFR 119…201 IVNANWKTAGEQSAADGFHT-LTLHRWLGE 22983 GHLNAORHRGMQVCRAEMGNAS-HFROPYHGWTYS 116…198 VVDANWKLGADNFVGDAYHT-MMTHRSMVE 22676 AFLNVORHRGKTLVNAEAGNAK-GFVOSYHGWGFG 109…190 VIKANWKAPAENFVGDAYHV-GWTHASSLR 21876 AFLNVORHRGKTLVNAEAGNAK-GFVOSYHGWGFG 109…190 VIKANWKAPAENFVGDAYHV-GWTHASSLC 21876 AFLNVORHRGKTLVSVEAGNAK-GFVOSYHGWGFG 109…190 VIKANWKAPAENFVGDAYHV-GWTHASSLR 21876 AFLNVORHRGKTLVSVEAGNAK-GFV0SYHGWGFG 109…190 VIKANWKAPAENFVGDAYHV-GWTHASSLR 21876 AFLNVORHRGKTLVSVEAGNAK-GFVOSYHGWGFG 109…190 VIKANWKAPAENFVGDAYHV-GWTHASSLR 21876 AFLNVORHRGKTLVHAEAGNAK-GFVOSYHGWGFG 109…190 IIKANWKAPAENFTGDAYHV-GWTHASSLR 21874 AFLNVORHRGKTLVHTEAGNAK-GFVOGYHGWGYG 107…188 VVKANWKPFAENFVGDTYHV-GWTHAAALR 21679 AFLNVORHRGKTIVDAEAGNAK-GFVOGYHGWGYG 112…193 VVKGNWKVFAENFVGDTYHI-GWTHASILR 22174 AFLNVORHRGKTLVHAEAGNAK-GFVOSYHGWGFG 107…188 VIKANWKAPAENFVGDAYHV-GWTHASSLR 21677 AFLNVORHRGARLCAVEAGNAR-GFAONYHGWAYG 110…191 FIEANWKAPSENFVGDAYHV-GWTHASALR 21981 AFLNVOTHRGARLVAAEAANAR-AFSOTYHGWSFG 114…194 LLNCNWKTPAENFVGDAYHV-GWTHLASLM 222

Amino Acid Sequence Alignments of Riske Iron Center Regions of Group 3 PAH and Terpine Dioxygenases

ConclusionsConclusions

� Degradation of xenobiotics in the rhizosphere is� Degradation of xenobiotics in the rhizosphere islikely enhanced by a combination of mechanisms,likely enhanced by a combination of mechanisms,including: fortuitous enrichment of degraders, including: fortuitous enrichment of degraders, growth linked metabolism, and cometabolism. growth linked metabolism, and cometabolism.

� Plant signal molecules including terpenes, � Plant signal molecules including terpenes, flavonoidsflavonoids, and , and salicylatesalicylate have multiple ecological have multiple ecological functions. When released into the rhizosphere,functions. When released into the rhizosphere,these compounds also have signal functionsthese compounds also have signal functionsfor inducing cometabolism of organic xenobiotics.for inducing cometabolism of organic xenobiotics.

Research NeedsResearch Needs

� Identification of specific plants that produce � Identification of specific plants that produce substances that can substances that can biostimulatebiostimulate xenobioticxenobioticdegradation.degradation.

� Evaluation of the role of plant, microbial, and � Evaluation of the role of plant, microbial, and earthworm enzymes (earthworm enzymes (oxygenaseoxygenase, cytochrome P450 �) , cytochrome P450 �) for effectiveness in degrading soil contaminants.for effectiveness in degrading soil contaminants.

� Studies on the ecology of rhizosphere microbial � Studies on the ecology of rhizosphere microbial consortia for enhancing bioremediation.consortia for enhancing bioremediation.

AcknowledgmentsAcknowledgments

Eric GilbertEric GilbertAndrew Singer Andrew Singer EkawanEkawan LuepromchaiLuepromchaiJoongJoong WookWook Park Park HaakrhoHaakrho YiYi

Development of Methods for Soil Bioaugmentation

Enzymes for Transformations of Monoterpenes