PlantPlant--facilitated Reductive facilitated Reductive DechlorinationDechlorination of of HexachlorobenzeneHexachlorobenzene in Constructed Wetlandsin Constructed Wetlands

The Third International The Third International PhytotechnologiesPhytotechnologies ConferenceConferenceAtlanta, GA, 2005Atlanta, GA, 2005

Xingmao Ma and John Pardue

Louisiana State University

AcknowledgementAcknowledgement

•• South/Southwest Hazardous Substance South/Southwest Hazardous Substance Research Center (HSRC)Research Center (HSRC)

•• John John ParduePardue, Han , Han WoongWoong Lee, William Lee, William MoeMoe

•• Stephen Stephen MbuligweMbuligwe, , ElaizaElaiza Alvarez, Maria Alvarez, Maria Murillo.Murillo.

AgendaAgenda

•• BackgroundBackground•• Experimental Setup Experimental Setup

and Analysisand Analysis•• ResultsResults•• ConclusionsConclusions

Background: Background: hexachlorobenzenehexachlorobenzene

• White crystalline solid

•Used as pesticides, fireworks

•highly oxidized

•Recalcitrant to biological process

•Reductive dechlorination is the only known biological process

Background: Reductive Background: Reductive dechlorinationdechlorination

•• Carbon and energy sourceCarbon and energy source•• CometabolismCometabolism•• DehalorespirationDehalorespiration

–– Low G+C Gram positiveLow G+C Gram positive–– ProteobacteriaProteobacteria–– Green nonGreen non--sulfur bacteriasulfur bacteria

•• DehalococcoidesDehalococcoides

Background: Background: DehalococcoidesDehalococcoides

Courtesy to Nature

Chlorinated contaminants

Reduced products

H2

electrons

ATP

H+

Strain CBDB1

Background: PhytoremediationBackground: Phytoremediation

•• A technology to use plants to treat A technology to use plants to treat contaminated mediacontaminated media

•• Many advantages over other technologiesMany advantages over other technologies•• Successful examplesSuccessful examples•• Less successful examplesLess successful examples•• PlantPlant--contaminantcontaminant--microbes continuummicrobes continuum

Experimental SetupExperimental Setup• 4L glass jars

• Artificially aged contaminants in sediments

• Two wetland plants

Typha

Phragmites

Materials and MethodsMaterials and Methods--agingaging

• Prepared 500 ppm HCB in diethyl ether (100mL)

• Added the solution to a 4L jar• Flushed away the solvents with pure

nitrogen• Added river sediment to about 65% of its

volume• tumbled it for 70 days

Materials and MethodsMaterials and Methods--samplingsampling

•• Sampled every 20 Sampled every 20 --30 30 daysdays–– 1010--15g for HCB and 15g for HCB and

metabolites analysismetabolites analysis–– 11--2g for microbial 2g for microbial

analysisanalysis

•• 2 planted reactors 2 planted reactors sacrificed at day 125sacrificed at day 125

Chemical analysisChemical analysis

•• 10 10 mLmL hexane/acetone (50%) addedhexane/acetone (50%) added•• Mixed 24 hoursMixed 24 hours•• Centrifuged at 3000rpm for 20 minutesCentrifuged at 3000rpm for 20 minutes•• Taken 1 Taken 1 mLmL supernatant for analysis with supernatant for analysis with

GC/MSGC/MS•• 10 10 uLuL internal standards addedinternal standards added

Results: HCB removalResults: HCB removal

0

2

4

6

8

10

12

14

0 25 50 75 100 125

Time (days)

HC

B (m

g/kg

)

TYphaPhragmitesControls

0

20

40

60

80

100

0 25 50 75 100 125

Time (days)

Per

cent

age

(%)

ControlTyphaPhragmites

ResultsResults--metabolitesmetabolites

Typha

0

0.005

0.01

0.015

0.02

0.025

0.03

0 50 100 150

Time (days)

Con

cent

ratio

ns (m

mol

/kg) HCB

1,3 DCB1,4 DCBM CB

Phragm ite s

0

0.01

0.02

0.03

0.04

0.05

0.06

0 50 100 150Time (days)

Con

cent

ratio

ns (m

mol

/kg)

HCB1,3DCB1,4DCBM CB

Microbial analysisMicrobial analysis--DNA extractionDNA extraction

•• SedimentsSediments--PowersoilPowersoil®® DNA extraction KitDNA extraction Kit

•• RootsRoots–– Washed with DI water and PBS washing bufferWashed with DI water and PBS washing buffer–– Lyophilized at Lyophilized at --5050ooC for more than 60hC for more than 60h–– Pulverized under liquid nitrogenPulverized under liquid nitrogen–– Extracted DNAExtracted DNA–– Stored in 100 Stored in 100 uLuL autoclaved DI water autoclaved DI water

RealReal--time PCR: Primerstime PCR: Primers•• Performed in Performed in iCycleiCycle PCR Detection SystemPCR Detection System

438438GGGAGTATCGACCCTCTCGGGAGTATCGACCCTCTCGGATTAGCTCCAGTTCACACTG GGATTAGCTCCAGTTCACACTG

DHC774FDHC774F##

DHC1212RDHC1212R

585585CCTACGGGAGGCAGCAGCCTACGGGAGGCAGCAGCCGTCAATTCMTTTRAGTTT CCGTCAATTCMTTTRAGTTT

U341FU341F**

B907RB907R

SizeSizeBase pairsBase pairsPrimerPrimer

* Casemayer et al. 2000. # Hendrickson et al., 2002.

RTRT--PCR: Temperature programsPCR: Temperature programs•• Reaction conditionsReaction conditions

7272ooC 6 minC 6 min7272ooC 6 minC 6 minExtensionExtension

7272ooC 1 minC 1 min7272ooC 1 minC 1 min

6060ooC 1 minC 1 min5555ooC 1 minC 1 min

9494ooC 30 sC 30 s9494ooC 30 sC 30 s

Reaction (x40)Reaction (x40)

9595ooC 3 minC 3 min9595ooC 3 minC 3 minDenaturation(x1)Denaturation(x1)

DehalococcoidesDehalococcoidesBacteriaBacteria

606000C 10sC 10s5555ooC 10sC 10sMelting curve (x80)Melting curve (x80)

Real Time Real Time --PCR: reagentsPCR: reagents

•• RTRT--PCR reagents (25ul reactions)PCR reagents (25ul reactions)

10.7uL10.7uLMolecular grade DI waterMolecular grade DI water

1.5ul1.5ulDNA templateDNA template

0.15uL0.15uL50 50 nMnM reverse primersreverse primers

0.15uL0.15uL50 50 nMnM forward primersforward primers

12.5uL12.5uLSYBR green PCR SYBR green PCR mastermixmastermix

Real time PCR: standardsReal time PCR: standards

•• Serially dilute known amount DNASerially dilute known amount DNA•• Standards for bacteriaStandards for bacteria

E. E. colicoli genomic DNA extracted from E.genomic DNA extracted from E. colicoli cellscells

•• Standards for Standards for DehalococcoidesDehalococcoidesE. E. colicoli plasmid DNA with plasmid DNA with Dehalococcoides Dehalococcoides 16S 16S rDNArDNAinsertioninsertion

•• DNA copy number calculationDNA copy number calculation

length660g/mol)10(6.023massDNA#

23

×××

=

Results: RTResults: RT--PCR standardsPCR standards

R2 = 0.9964

R2 = 0.9907

02468

10

0 10 20 30 40Ct

Logc

opy#

Eubacteria Dehalococcoides

Results: Microorganisms in the bulk Results: Microorganisms in the bulk sediment over timesediment over time

Dehalococoides in the Bulk Sediment

0.00E+001.00E+062.00E+063.00E+064.00E+065.00E+066.00E+06

0 30 50 90 125Time (days)

DN

Aco

pynu

mbe

r

Typha

Phragmite

Eubacteria in the Bulk Sediment

0.00E+002.00E+084.00E+086.00E+088.00E+081.00E+091.20E+09

0 30 50 90 125

Time (days)

DN

Aco

pynu

mbe

r

Typha

Phragmite

Results: Results: DehalococcoidesDehalococcoides/Bacteria /Bacteria in sediment over timein sediment over time

Dehalococcoides /Bacteria (%)

0.00.40.81.21.6

0 50 100 150Time (Days)

Typha

Phragm ites

control

Results: Day 125Results: Day 125

0.260.264.204.200.190.191.301.30Ratio (%)Ratio (%)

1.07x101.07x10663.27x103.27x10774.58x104.58x10552.87x102.87x1077DehalococcoidesDehalococcoides

4.14x104.14x10887.78x107.78x10882.35x102.35x10882.16x102.16x1099EubacteriaEubacteria

sedimentsedimentrootsrootssedimentsedimentrootsroots

PhragmitesPhragmitesTyphaTyphaItemsItems

ConclusionsConclusions

•• Both aquatic plants facilitate reductive Both aquatic plants facilitate reductive dechlorinationdechlorination of HCBof HCB

•• Plants increased the bioavailability of aged Plants increased the bioavailability of aged contaminantscontaminants

•• RTRT--PCR is a useful technique to quantify specific PCR is a useful technique to quantify specific microbial groupsmicrobial groups

•• Plants selectively enriched certain Plants selectively enriched certain microorganismsmicroorganisms

•• Plant roots provide locus for Plant roots provide locus for dehalorespringdehalorespringbacteria except for exudates stimulationbacteria except for exudates stimulation

QuestionsQuestions

Questions???Questions???

RealReal--time PCR: backgroundtime PCR: background

Quantitative PCR

DNA binding dye

PlantPlant--facilitated facilitated TeCBTeCB degradationdegradation

0.0000

0.0040

0.0080

0.0120

0.0160

0.0200

3 5 7 9 11 13 15 17 19 21 23 25 27 29

Time (d)

TeC

B C

once

ntra

tion

(mol

/kg)

Control Phragmites Typha

Hydrogen and Methane ProductionHydrogen and Methane Production

02468

10121416

0 3 6 9 12 15 18 21 24 27 30Time (d)

H2,

aqu

eous

co

ncen

tratio

n(n

M)

Control

PhragmitesTypha

0

0.4

0.8

1.2

3 7 11 15 19 23 27Time (d)

CH

4ar

ea(x

106 )

ControlPhragmitesTypha