aerobic and anaerobic biodegradation of pcbs · aerobic and anaerobic bipdegradation of pcbs •...
TRANSCRIPT
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Aerobic and Anaerobic Bipdegradation of PCBs• • ' /= . ,
Presentation toEPARegionll
Daniel A Abramowicz, ManagerEnvironmental Technology Program
Biological Sciences LaboratoryGE Research & Development Center
New York City, NYDecember 17,1990
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Aerobic Degradation• Introduction• rDNA Efforts• Dragstrip
Anaerobic Dechlorination• Introduction• Aroclors• Single Congener
Anaerobic/Aerobic• Lab Results
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anaerobicbacteria
aerobicbacteria
cells+
CO2+
H2O
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fte,
5'•v <r 6"
14'-biphenyl [95-92-4]
2,3,3'»4,4'-pentachloro,l ,1 '-biphenyl2,33',4,4'-pentachlorobiphenyl
234-34
OFfliO (i0-)meta (m-> 1,3-para ^p-) 1,4-
o-
HI-
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RELATIONSHIP BETWEEN NUMBERS OF ORTHO AND NON-ORTHOCHLORINE ATOMS IN COMMERCIAL AROCLORS
Figure 8-L
i 2 3 4NUMBER OF ORTHO CHLORINE ATOMS
Relationship between numbers of ortho and non-ortho chlorine atomsin commercial Aroclors.
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f *
7:1" ii0</>
4-
1
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Cl,CL Cl,
CI4 Cle•••••••••••MHB • • •• *«MMBBIMm
cu
1
JuU
AROCLOR 1260
CLCl, Cle
AROCLOR 1221
jjjAROCLOR 1242
AROCLOR 1248
uu
AROCLOR 1254
Cl10
Cl
AROCLOR 1268
CIC!3 .
CI4 _CjeJ_"~~CI5
ciLAJL
C CI
A
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Table 1: Identification of PCB'degrading environmental isolates
Sfrain designation
MB1Pi434
H201,Pi704,RJBHI 28, H336, H430LB400, LB410Pi939, HI 130, Pi304
H702, PilOl
amsm
Corynebacterium sp.Alcaligenes faecalisAlcaligenes eutrophusPseudomonas cepaciaPseudomonas testosteroniPseudomonas sp.Pseudomonas (Acidovorans group)
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Bacterial Strain
PCIcongener
o100>0000
2,32,4'
4,4'2,4,4'2,5,4'2,*,3',4'
2,2'*,3,2',3'2,9,2"2,3,2',5'
2,4,5,2',3'2>4,2',4'
2,3i*,2',5'2,4,5,2',5'2,4,5,2',4',5'
"3- CO O>O Q CO« #.. g>Q- A.,0k
CM <? g JT
.g ':£ Es 5 S
•• • • •• •
o>« H .Jk. ft.
"" « "* S 3j 5: a: z ui i
• ••••• • . •
99 •• ' •
• • • • *
• •
*«-it•v
o oO «- Ox» * *S 2 S
o • •
Figui-e 1: Comp«s0fl ^ ti^ PCl-4^rad^ competence of environ-me^tal bacterial kdla|g$/ [0] Wfei^s ^^ H850 degraded less than20% of this conger (243U245-CB), bat a iti^abolite was isolated.[adapted from
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Biphenyl and PCB Biodcgradation Pathway
C02H
oto
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H850/LB400
MB1/H850/LB400
a
Figure 3: Initial oxidative degradation products for attackvia 2,3- and 3,4- dioxygenases on the congener 2,3,2',5'-tetrachlorobiphenyl.
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A)
B>
uC)
D)
LB400
MB1
I3 4
LB400 and MB1
Figure 4: Biodegradation of PCBs on soil by LB400 and MB 1(SOppm Aroclor 1242, 109 cells/0.4 gm soil) Shaded peak isnot degraded (246-24-CB) and is used as an internal standard.[adapted from reference 4]
Kfcl *i-
otoo\ri^to
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Aerobic Degradation• Introduction• rDNA Efforts• Dragstrip
Anaerobic Decfalorination• Introduction• Aroclors• Single Congener
Anaerobic/Aerobic• Lab Results
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1
SCISSORS
PLASMIDpSCOl
RECOMBINANT DNAMOLECULE
INTRODUCTIONINTO HOST C€Ll
JOINING
FOREIGN DNA TOBE INSERTED
ANTIBIOTIC -RESISTANCEMARKER
SELECTION FOR CELLS CONTAINING RECOMBJNANT DNA MOLECULESBY GROWTH IN THE PRESENCE OF ANTIBIOTIC
Figure 5-11The cloning of DNA in a plasmid.
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A)bph A COOH
B)
oto
CJ1
CC 1~ o(A O
CO UJ I
2.0 6.1
DC CC CC CCO O 0 0O O O ojj UJ UJ UJ
0.8 2.0 2.9 6.65| ....bph A.........bph B/C...
CCOj0
Uii
, , , l
CC00
UJ
2.3 0>ph D
OC
I s !l U J l
.5
IPCB METABOLISM
Figure 2: A) Metabolic pathway for biphenyMPCB degradationB) Arrangement of the EcoRJ fragments in pGEM410
bph A - biphenyl/PCB dioxygenase Jbph B - dihydrodiol dehydrogenasebph C - dihydroxybiphenyl oxygenasebph D - hy<ir»se
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AUHoo
B
I J. -AA/-j UL)Lj
LUFigure 4-3. Degradation of Aroclor 1242 (10 ppm) by PUudomonat strain LB400
and K coU FM4560 in a 24-h resting cefl assay. Panel A, LB400;panel B, FM4560; panel C, nercuiy-ldlled control.
38
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TaMt4-2
f ACTtRlAL STRAINS
RGBCONGENER
~ e< & *
s:K SI "» ° * «0 «
o » w « 2 w f l » « w l l r t ^llrtlf2 » - « 2 » - ^ r » - » - S « ~ 2l«ltnX u i x a . x x u i x 2 E x c L l x | 3 B56
2,32,4'
4,4'2,4,4'2,5,4'2,4,3',4'
2,2'2,3,2',3'2,5,2'2,3,2',5'
2,4,S,r,$'2,4,2'f4'2,5,3',4'3,4,3',4'
2,S,2',r2,3,4,2',S'2,4,5,2',*'2,4,S,2',4',r
ffttffftft000*000000000000000000000000000000000*000000• ••000000000000000000••00000••000000000««
000000
•••00000000000000• 00••0000• * • 9000000
0**0000
KEY:% DEGRADATION•20-39• 40-59
60-7980-100 000
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Table 2: Relative PCB degrading activity of E. coli recombinantstrains an&Pseudomonas strain LB400.PCBCongener
2.3
2.4'
2,5,2'2,5,4'2,3,2',3'2,3,2',S'2,5,3',4'
1,4,*,*',*'
4,4'.4.4*
.4.2X,4,3',4'
Percent Activity *LB400
(Blphenyl)FM
4100FM
45COLB400
<S)
'Indicated »» UM percent of degradattoncompared wKh Wphenyl- grown LB40AResting cell assay: Mix IB, 2BFNT4 1 00: H8 1« 1 (pGEM4 10)
LB40D (S): grown using succkiate
Key: % Degradation»t-180ft-7t
• 4t-ft* 2t-ltO 1-1»
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itH(tter3*xAlaIl*
101 i'OBTcaTCMauuyu>uu*j'i<XTT6*TccAoocA 200eClMUallaJUr^
201 TCTACCCCCATCACAt. JCU lATCAQCTOGAOCTTCAQC.'COUl HI lUaOOClC H.rTACTTOOaCJICCJ>CMTCAICTOCCTC*AACCOOOCAU«LMt61yU*eiBte(^
401la^
sai «o«>»lj l
601 700
701 800
B01 AC
901 MODCly»jci<jr* ltto^
looi 1100
1101 TCCCCOTCTC<XaCCCGCXaCCTCCCAATCAAATCGl>GStGrC<ifcX.Cl 'lUKXCTGSICGAT ATCAAGCAAGAATATCGCCG 1200
1201 «ACAACATCOX»ACTTCTCC«»«X:«JCCTCTTTGA<XAG 1300
1301 ACgCACCCGCTCAATCCCCAGATCGCXXilCCCTOtglCOCACAOCCGTCTCCCTCai'll'l ^ 1400SerGlnProLouAsnAlaGlnMetGlyLeuGlyArgSerGlnThrGlyHlsProAspPheProGlyAsnValGlyTyrValTyrMaGltXSluAlaAlaA
1401 G«arraTCTAtCACCACTG«TGCGCAT<»TGTCqa<XC«^ 1500rgGlyMetTyrHlsHlsTrpMetArgMetMctSerGluProSerTrpAlaThrI«ul.y»ProEnd
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DC
& 1=ip
1Prebe 1
Probe 2
410
otoa\eno
CO
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, - \
f Cfc dc
15 kt
Ws *
vM
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Aerobic Degradation• Introduction• rDNA Efforts• Dragstrip
Anaerobic Dechlorination• Introduction• Aroclors• Single Congener
Anaerobic/Aerobic• Lab Results
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T f
;• ••&>!:'-.•./i'-vV/--.-'.
'It> v;'.-*i;''/--
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.•'
'.. '''•.'••" ' • ' • ' - -'' -:'^S^' " ' '
PCB EXTRACTION FROM OLENS FALLS SOft.RQURE1: KINETIC DATA - SDBS ON TWO SOILS
oIcoOCL
3;CO
1% SOBS ON OAKLAND SOIL @ 80 C
1% SDBS ON GLENS FALLS SOIL© 80 C
EXTRACTION TIME
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PCB EXTRACTION FROM GLENS FALLS SOILFIGURE 4: EQUIL PARTITION CURVES FOR 1 % SOBS SOLN
8 *1" "*
:••••:.:
UNTREATED QLENS FALLS SOILKpav««5 5.8 Vkg
CAUSTIC TREATED SOIL
CAUSTIC & HEAT TREATED 9C*.Kp «v« = 0.9 I/kg
SOt'N PCS CONCEWTIWDON
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Aerobic Degradation• Introduction• rDNA Efforts• Dragstrip
Anaerobic Dechlorination• Introduction• Aroclors• Single Congener
Anaerobic/Aerobic• Lab Results
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•* I
x *
Ln n
i i i i i iJL fc.^ t^ -JL. - - . -J.-A__
k i
JUi JU.U
X 1ill II I I I
Fig. 1. DB-1 capillary' gas chromatograms (plots of detector ftspomc versus eiution time) of WpcrHudson River sediments that show surface pattern A (largely wncfcangcd Aroclor 1242) and subsurfacepatterns B, B , ancUC. Aflame ionization detector was wsed so Aat ate PCS peak response was nearlyproportional to molar concentration; however, BOB-PCB iwpHrincs in the samples also produce*!observable peaks (designated * and Imp.). The major Pd congeners KS|>onsibk for the observes* ecafcsare designated by the numbers that correspond to Ac poodoa rf cMomes on each of Ac two pftcwrtrings; thus 2-2 and 24-4 indicate 2,2'-<8chlorobiphcByl and 2,4,4'-«ichk>robiphenyl, respectively.Internal, standard peaks are designated Int. std.
B-B' and C were associated with the deeper"hot spots," which have been estimated tocontain 77 metric tons of PCBs (6).
Quantitation of the individual capillaryGC peaks indicated that the levels of most
1 tri- and tctrachtorobiphcnyls were depressed.relative to those in Arodor 1242 in aflclasses of upper Hudson River sediments,but particularly in those that showed pat-terns B, B', or C Summary data for 2,5,4'-plus l '-dvloro&phenyl (CB) and for2,5,3',4'-OI (which arc representative ofcongeners with leaser or greater rcsponsivc-hess «o dechtorinadon. respectively) areshown in Table 1. Convener/, in all sedi-ment classes the kveb of the 2,6,2'* and2,6,3'-CBs and those of all dkhlorobiphcn-yb were increased two- to sixfold, and thelevels of the monochlorobiphcnyl 2-CB in-creased 7- tp- iTlold, with the largestchanges ot^crvcd in the samples thatshowed patterns B, B', cr C (Table I). Theincreases in die mono- and dkkbrobifteB-yls occurred despite their fyeaeer tendencyto dute into Ate fiver water or wxtogoacrobk l)iodcgradatioa. Tkus k was cfvideacthat in the upper Hudson River as a wfeofc amassive (40 »o 76 metric tons) coBverskM oftri-, tetr>, and higher ehloK>biphe»yJs tomono-, «S-, aod 2,6,X'-tFichJorobig>henyls(X' = 2, 3, or 4) kad occurred, paniculariyin die subsurface (15- to 30-year-old) por-tiofi of tike sediments.
The sediments of Silver Lake, a 1-0-haurban pond ia PittsfieW, Massachusetts,eootaia an estimated 29 metric tons of PCBs(iff), wisach are believed to have originallyteen almost entirely Arodor 1260 releasedfrom adjacent transformer-manufacturingoperations before 1972. A mapping and
Arocloriaeo
_>—IL-.
Pattern F («G)
710
Fig. 2. DB-1 capillary gas chro-matograms (plots of detector re-sponse versus eiution time) of Aro-clor 1260 and of the Aroclor 1260residue extracted from a|SUve£Lakesediment composite that showedmamly^pattem F (with some .pat-tern G, which contributed the threesmall peaks on the left). These chro-matograms were obtained with anelectron capture detector. Such de-tectors give a stronger responsewith the more heavily chlorinatedPCB congeners, a weaker responsewith the less heavily chlorinatedones, and little or no response withunchlorinated impurities. The ma-jor PCB congener peaks and theinternal standard are designated asin Fig. 1.
SCIENCE, VOL. 236
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rate** of (Sl-Ws V dxAfr*M*«W S««K*€.t&C *,
RELATIONSHIP BETWEEN NUMBERS OF ORTHO AND NON-ORTHOCHLORINE ATOMS IN COMMERCIAL AROCLORS
Figure &-1.
. 1 2 3 4
NUMBER OF ORTHO CHLORINE ATOMS
Relationship between numbers of ordw and iwi\-ort/io chlorine atomsin commercial Aroclors.
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Comparison of Environmentally Transformed PCBand Laboratory Incubation of PCB
Aroclor 1242
ICM
NI
C4
M
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MI
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Environmental Transformationof PCB
Hudson River Sediment
CMI<a
CMIto*
CM
Anaerobic IncubationArocJor 1242/Hudson River
Sedkeemit16 weefc^
402662
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Aerobic Degradation• Introduction• rDNA Efforts• Dragstrip
Anaerobic Dechlorination• Introduction• Aroekwrs
Se Congener
Lab Results
402663
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Figure 6-2: Dechlorination patterns observed under different conditions after 18 weete.Panel A, autoclaved control; Panel B, includes RAMfM (pattern M); Panel C, includesRAMM + cysteine hydrochloride at 1 gm/L (pattern Qj). • as-
402664 an-
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402665
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ABSOLUTE DIFFERENCE(MOLE PERCENT) MOLE PERCENT
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MOLE PERCINT
o o o e & gMOLE PERCENT
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Water-only Addition9
Aroclor 1242/1254/1260
80 100
4.5
1.5
-1.5
-4.5
-7.5 'iimiiuH
Difference
20 40 60 80 100
Peak Number in DB1 (118-peak profile) 402668
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Nutrient Acceleration
£o2
Aroclor 1242/1254/1260
4.5
-7.5 ^0
80 100
20 40 60 80 100
Peak Number in DB1 (118-peak profile)402669
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of ais of
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sev-ium-iGCs ofjqualsrinesities
Figure 2. Gas chromatogram of Aroclor 1242 on SE-30 withan electrolytic conductivity detector. The\peak identificationnumbers correspond to the retention tirr^ relative to p,p'-
slQQ. From in^ctten, at the arrow, to peak 146 wasabout 20 min.
OMCTk<l01
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Billings Island(Griffin Island)
Village ofFort Edward
*- Rogers Island
H7 site(1991 field test)
PC6
Thompson Island
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Widespread Anaerobic Dechlorinating Microorganisms:
Contaminated Sediments
Escambia Bay, FL /Hoosic River, MAHudson River, NYNew Bedford Harbor, MASheboygan River, WISilver Lake, MAWaukegan Harbor, ILWoods Pond, MA KflU*a*>>Mx
Utoeontaminated Sediments
Adirondack Muck, NYCeotei Pond, MARed Cedar River, MISaline River, MISpier Falls, NY (Hudson River)
402677
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Mole Percent Mole Percent
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Endogenous Dragstrip PCBs
£o
o3s
Experimental ow
100
-7.540 60
Peak Number in DB1 (118-peak profile)100
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Endogenous Hudson River PCBs 0*7)
15
12
20 30 40 50 . 60 . X 70
12
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Difference
20 30 40 50 6lPeak Number in DB1 (118-peak profile)
402680
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PCB CONGENER DISTRIBUTIONIN WOODS POND SEDIMENT
BEFORE AND AFTER DECHLORINATION1
IllOccUJCLLU
10 ' 20 30 40 50 60 TO
ZUJOECLUEL
0
DAYS
ifliltlpiHfMf liill1 50 ' 60' 70T 00
jrHTnrryni \ \\ uii t H H i mmTiTiTiii mb\wm iiitli ini30 * 40 ! 50 ' 60 ' 70
GC PEAK NUMBER
1SECOND TRANSFER, 23456-CB, 69-5A1-133
idybnlfi© '' tf0 '
402681
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Aerobic Degradation• Introduction• rDNA Efforts• Dragstrip
Anaerobic Dechlorination• Introduction• Aroclors» Single Congener
Anaerobic/Aerobic• Lab Results
402682
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WoodsPond
SilverLake
HudsonRiver
96%
14%
63%
3%
79%
34%
1%
7%
3%
402684
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Concentrations of Planar PCB Congeners (Dioxin-like toxicity)
Congener* Cone, of Congener in Cone, of Congener in %Aroclor 1242 Dechlorinated** Reduction
f ^ a\ Aroclor 1242* *• Jk - / XV/ \ / f+4 \
34-34345-34
345-345245-34234-34
2345-34
0.140.0030.00150.330.430.008
< 0.0050.0005
< 0.000150.0680.0610.0043
>96.5 -83
>90808646
Most active congeners in AHH/ERODen2yme indication assaysMicrobial dechlorination pattern C withHudson River sediments \
IOtoa\ooo\
EROD assay demonstrated 75% reduction in enzyme inductionwith dechlorinated Aroclor 1242
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Transfer Experiments onto Different Supports
#C1 removed/25 wks
No support 0.0Sand 0.0Sawdust 0.0Clay 0.0Vermiculite 0.0
Whole sediment 1.3Peat 0.6Peat/Vermiculite 1.2Peat/Clay 0.5
402687
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RELATIVE PERCENT RELATIVE PERCENT
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10 21 38(Celsius)
402689
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X Conversion to 26 CB X Cofwtwiw to 26-CB
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OtoO^IDto
EFFECT OF INCUBATION TEMPERATUREON DECHLORINATION OF 236-CB IN
CULTURES OF WOODS POND SEDIMENT
O 24°C30°C
D 16°C
10 15 20 25 30 35 40 45 50 55 60 65 70 75INCUBATION TIME (DAYS)
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Aerobic Degradation• Introduction• rDNA Efforts• Dragstrip
Anaerobic Dechlorination• Introduction• Aroclors• Single Congener
Anaerobic/AerobicLab Results
402694
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BACTERIAL TRANSFORMATION OF PCBs
Organisms
Location
Requirement forActivity
TypedTransformation
Best Stubstrates
AEROBIC BACTERIA
Pseitdomonas, Alcallgenes,Corynebacterium,...
Water Column,Surface Sediments
Oxygen
OxidativeRing Cleavage
MofK> toPeMa-CB
ANAEROBIC BACTERIA
Unknownx
Sub-sediments
Lack of Oxygen
ReductiveDechlorination
Tri-toOcta-CB
402695
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y**w»|X,
O.
tO O
ttl
o o o o
1J
o
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500
h- 400
200
monooto
di
• Control 1242/1254/1260(H Anaerobic Dechlorination (Pattern Q)• Anaerobic + Aerobic (LB400/MB1)
tri tetra perrta hexa hepta octa nona decaHOMOLOG GROUP
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PCB CONCENTRATION (fJLM)ro £» o> oo o K>o o o o o o
!•
lAf
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/—^
402699
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xi
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oto
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r\
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402703
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Estimated Time Schedule
March 1,1991
April 1,1991
May 1,1991
June 15,1991
September 15,1991
October 15,1991
Final hardware design andexperimental plan
Hardware assembly begins atfabricator shop
Site preparation begins
Site preparation completedHardware assembly completedConstruction of facility begins
On-site construction completedIn situ test begins
In situ testing completedSite closure begins
Site closure completed
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Estimated Protocol
OH 1: Isolation only (control).
Cell 2: Nitrogen sparging and rake agitation only (control).No nutrient or microbial additions.
Cell 3: Full process control with added naturalmicroorganisms cultured in the laboratory (e.g., pH,temperature, oxygen level, nutrient composition,agitation, and cell density).
Cell 4: Full process control with added indigenousmicroorganisms from the site cultured in thelaboratory (see Cell 3).
Cell 5: Partial process control with added naturalmicroorganisms (e.g., ambient temperatureand pH, limited nutrients, agitation,aeration, and cell density).
Cell 6: Partial process control with indigenousmicroorganisms (see Cell 5).
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Drawing, shows proposed lo-by-25-foot experimental station to be placed in Woods Pond inLenox. rond is actually 230 acres, and trie final project may include a 30-foot ramp fromshore.
, WO
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Important New Findings;
- Rates at lower temperatures ~room temperature rates
- Transfer/enrich organisms
- Organisms are common• Contaminated sediments• Uncontaminated sediments
- Potential for complete anaerobic degradation• Hudson River organisms can remove all outer
chlorines• Housatonic River organisms can remove inner
chlorines
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SUMMARY
• PCBs do biodegrade• widespread activity in sediments• aerobic• anaerobic• [PCB] in sediment/bass
• Attractive alternative• in-place• natuiai process• less invasive
• Moving forward• accelerate this natural process• lab scale• river model• site test in the river (1991)
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PCB PROGRAM PERSONNEL - HISTORICAL LIST
/****K
/**"T\
AEROBIC DEGRADATION
Organism isolation:Donna L. BedardLawrence H. BoppMichael J. Brennan, Jr.Cart JohnsonJohnH. LobosRonakJ Unterman
Biochemistry/Pathways:Donna L. Bedardlawrence H. BoppMichael J. Brennan, Jr.John F. Brown, Jr.Marie L HaberlRalph J. MayRonald UntermanRobert E. Wagner(University of Iowa)(University of Kentucky)
Application/Scate-up:Angelo A. BraccoRonald E. BrooksKenneth M. CarroflDavid K. DietrtchMark R. HarknessJohn B. McDermottDavid P. MobleyCharles SchwartzGregory L. Warner
Molecular Genetics:Bruce D. EricksonFrank J. MondeDoJames R. Y&tes
Metabolism:John A. BergeronBruce D. EricksonKenneth M. FishDavid W. KruegerDavid T. Lin
ANAEROBIC DECHLORINATIQN
Hudson River:Daniel A. AbramowiczMichael J. Brennan, Jr.Edie L GallagherChitra StokesHeidi M. Van DortWilliam A. Williams(Michigan State University)(New York Univ. Medical Center)(Oregon State University)(Stanford University)(State Univ. of New York, Syracuse)(University of Georgia)
Woods Pond:Donna L. BedardStephen C. BunnellHeidi M. Van Dort
Models/Scate-up:Mark L Stephens(Ceigene Corporation)(University of Michigan)
ENVIRONMENTAL ASSESSMENT
John F. Brown, Jr.George M. Frame, IIRalph J. May(AccuStandard)
Oto•~JOvo
7/90