Technologies for Biogeochemical and Hydrogeologic Characterization and

Their Integration for Site Remediation

Michelle M. Lorah U.S. Geological Survey MD-DE-DC Water Science Center mmlorah@usgs.gov

1

ers

• Complex hydrogeologyhydrogeology • fractured rocks • low permeability

la rock matri layers; rock matrix

• Difficult contaminant mix • DNAPL • dissolved and

highly sorbed mixhighly sorbed mix • emerging

contaminants • Sensitive habitat or • Sensitive habitat or

location • wetlands • bottom sediments

2

Biogeochemical Characterization-Why?Why?

• Provide the remedy-MNA, bioremediation,, , biogeochemical reduction

• Secondary effects-alteration of naturalalteration of natural biogeochemical conditions, or from presence of secondary contaminantsy

• Long-term efficiency • changes in transmissive

plume with remediation p

• low permeability zones

• “slow” processes key (back diffusion, sorption( p and desorption, abiotic and biotic degradation reactions) 3

tools

-

Biogeochemical Characterization-H ?How?

Removal

• MNA protocols provide good framework and tools

of contam-

inant

• Relevant protocols for organics, radionuclides, and non-radionuclide

Geochem indicators and non radionuclide

inorganic

• Three lines of evidence for chlorinated solvents

(DOC,, redox, metabolites,

pH, Cl )

for chlorinated solvents

Microbial ti itactivity

4

Considerations

• History and stage of plume evolution

DNAPL LNAPL• DNAPL or LNAPL presence

• Sample key parts of l l dplume, including

“transition zones”

• Multilevel sampling-high resolution

• Spatial and temporal variability

• Interaction with and formation of solids

from Monitored Natural Attenuation of Inorganic Contaminants in Ground Water,Volumes 1 and 2 5

O2

dissolved plumes

Aerobic micro-zones around roots

Fe2+ S2-

NH3

CH4

DNAPL

O2

WETLANDSWETLANDS LARGETRANSITION ZONELARGE TRANSITION ZONEWETLANDSWETLANDS-- LARGETRANSITION ZONELARGE TRANSITION ZONE (MODIFIED FROM(MODIFIED FROM LORAHLORAH ET AL., 2005)ET AL., 2005)

Stottmeister et al.(2003) Biotech.Advances 6

a e Co a a s

Canal Creek Area Aberdeen Proving Ground Canal Creek Area, Aberdeen Proving Ground

Chlorinated VOCs-Anaerobic Anaerobic degradation

Parent Contaminants

Chlorinated ethanes: HCA= hexachloroethane PtCA= pentachloroethanePtCA pentachloroethane 1122TeCA= 1,1,2,2­tetrachloroethane

Chlorinated ethenes:Chlorinated ethenes: PCE= tetrachloroethene TCE= trichloroethene

Chlorinated methanes:Chlorinated methanes: CT= carbon tetrachloride CF= chloroform

7

1122-TeCA plume

W B h C l C k N l A i S d A RWest Branch Canal Creek, Natural Attenuation Study Area: Reddox

Methanogenic

8

West Branch Canal Creek Natural Attenuation Study Area: VOCsWest Branch Canal Creek, Natural Attenuation Study Area: VOCs

VC

C

1,1,2,2-Tetrachloroethane (µg/L)

12DCE 12DCA

TeCA 2.0μM, 90cm

9

Seep Areas

10

West BranchWest Branch Canal Creek

Deggradation in non-seep areas where relatively slow flow allows strongly reducingg conditions.

NON-SEEP AREA SEEP AREA

Ammonium

Methane

Fe(II) S2-

Fe(II)Methane

PCE

CT

CF

VC

Upward 12DCE

12DCA

flow TeCA 2.0μM, 90cm

11

g

MNA WBC2

WBC-2 Dechlorinating Consortium, developed to degrade 1,1,2,2-tetrachloroethane (TeCA)

Manchester, et al, 2011

1 000

10,000

0 60

0.70

0.80

/TeC

A)

100

1,000

0.30

0.40

0.50

0.60

Hal

f-lif

e(da

ys)

on (D

augh

ter/

VC

tDCE

cDCE

1

10

0.00

0.10

0.20

CC Aquifer CC Wetland (23)

CC Wetland (30)

CC Aquifer CC Wetland (23)

CC Wetland (30)

TeCA

H

Mol

e Fr

acti

o cDCE

TCE

12DCA

112TCA (23) (30) (23) (30)

TeCA

TeCA t1/2 WBC-2MNA 12

h d

y

Standard Chlorine of Delaware, DNAPL Extent

CB DCB TCB CB, DCBs, TCBs DCBs, TCBs Not indicative of DNAPL

USGS Study Wetland

characterization

Wetland porewater

characterization Natural

attenuation;

Aquifer

enhanced bioremediation

Feasibility of permeable reactive barrier

Containment wall

13

11221122--TeCATeCA

PCEPCE

112TCA

TCETCE CTCT

112TCA

12DCE CFM

A b ( d )12DCA

VC MeClCO2

u as a

CA

VC MeCl -

Ethane Ethene Methane

PCEPCE135TCB,124TCB, 123TCB

Chlorobenzenes-Standard Chlorine

Trichlorobenzenes*

of Delaware

Dichlorobenzenes* Anaerobic (reductive

dechlorination)

70 (124TCB)

14DCB, 13DCB, 12DCB Dichlorobenzenes*

Aerobic CO2, HCl

dechlorination) • CB serves as terminal

electron acceptor • Separate e- donor 75 (14DCB)

Chlorobenzene* HClp

required • rate decreases with

decreasing number Cl

Benzene *

?? Aerobic (oxidation) • O2 required as

electron acceptor • CBs tilized C nd

100

CO2 CH4

• CBs utilized as C and e donor

• Short-lived intermediates

5

D i ki W t CO2, CH4 * Parent contaminantParent contaminant

• rate increases with decreasing number Cl

Drinking Water MCL µg/L

14

20.0

25.0

gram

s pe

r

Northwest PDBs, October 2011 Methane

Sulfide

10.0

15.0

atio

n, in

mill

ig

liter

Sulfide

Ferrous iron

Ammonia

5.0

Conc

entr

a

0.0 8 102 131 132 133 134 135 11 138 139 140 141 142 143 144

Site Number 40.0

per

Northeast PDBs, October 2011 Methane

25.0

30.0

35.0

n m

illig

ram

s p

er

, Methane

Sulfide

Ferrous iron

10.0

15.0

20.0

cent

rati

on, i

n lit

e

Ammonia

0.0

5.0

106 107 108 109 110 6 112 113 114 14 116 117 118 119 120 122 123 15 125 126 127

Con

Site Number 15

9 8 1 2 3

V

25

30

35

80%

100% Peeper Site DP-8 (NW)

Benzene

SCD, VOCs in

15

20

25

40%

60%

VO

Cs (m

g/L)

Mol

ar p

erce

nt CB

12DCB

13DCB

14DCB

Peepers

0

5

10

0%

20%

2.0

7.5

3.1 .6 4.1

9.7

5.2

0.7

6.2 .8 7.3 .8 8.4 .9 9.4

5.0

0.5

6.0

1.5

7.1

Tota

l M 14DCB

TCBs

Total VOCs

50 90

100

Upland Wells, Oct . 2011

2 7 13

18 24

29 35

40

46

51 57

62

68 73

79 85 90

96 101

107

Depth below land surface, cm

20

30

40

40 50 60 70 80

al V

OCs

(mg/

L)

ar p

erce

nt

0

10

20

0 10 20 30 40

Tota

Mol

a

16

4 7 4 7

Iron, Sulfide- Sediment Cores

0 100 200 300 400 500

Concentration, µmol/g

Feet bls 0 100 200 300 400 500

Concentration, µmol/g

Feet bls

0.5-1.0

1.4-1.7

6

Fe2+

Site 8 (NW)

Sit

0.5-1.0

1.4-1.7

2 3 2 6

Fe2+

Site 107 (NE)

Site 6 (NE)2.3-2.6

0.5-1.0

1.4-1.7e3+

Site 104 (NW)

2.3-2.6

0.5-1.0

1.4-1.7e3+

( )

2.3-2.6

0.5-1.0

Fe

2.3-2.6

0.5-1.0 Fe

1,200

1.4-1.7

2.3-2.6

S 1.4-1.7

2.3-2.6

S

17

-

ToolsTools toto EvaluateEvaluate BBiodegradationiodegradation Molecular Biological Tools GEO• Molecular Biological Tools • Quantitative PCR- Counts genes,

taxonomic or functional, for specific t t i (Q tA MI)

GEO anions, VFAs

COC targets; micro-arrays (QuantArray, MI)

• Terminal Restriction Fragment Length Polymorphisms (TRFLP)-fingerprint of the microbial

COC VOCs, redox

MICRO fingerprint of the microbial community

• Next-generation sequencing (high throughput)- in depth profile of the

MICRO Bio-sep beads-microbes throughput) in depth profile of the

microbial community; Illumina, 454 sequencing

• Stable Isotopes

and 13C Amendments donor • Stable Isotopes

• SIP, Stable Isotope Probing- 13C used as a tracer

CSIA C d S ifi I t Bio-Sep® beads provide a large

Bio-Traps, Microbial Insights

• CSIA, Compound Specific Isotope Analysis- isotopic fractionation in parent and metabolites

provide a large surface area for microbial attachment

18

In situ microcosms with BioIn situ microcosms with Bio--Traps (Microbial Insights)Traps (Microbial Insights)

T h h d• Two each northwest and northeast sites

• Three standard treatments and three 13Ctreatments and three 13C-labeled treatments

• MNA, monitored natural attenuationnatural attenuation (no amendments)

• Lactate, biostimulated with

3 0 Site 107 Redox

lactate +chitin • WBC-2,

bioaugmented2.0

3.0

on (

mg/

L)

Fe2+ Sulfide Methane

8.6

• 13C-labeled chlorobenzene

• QuantArray analysis of i d f i l0.0

1.0

Con

cent

rati

o Sulfate

species and functional genes for aerobic and anaerobic biodegradation

0.0 MNA Lactate WBC-2

C

19

o

ISM 1.00

1.20

mol

/L) Standard ISMs, DP6 14DCB

13DCB 12DCB

Results: 0.60

0.80

trat

ion

(µm 12DCB

CB Benzene

•Complete

0 00

0.20

0.40

Conc

entComplete

degradation of DCBs evident in WBC-2 treatment

0.00 MNA Lactate-Chitin WBC-2in standard ISMs

•13C-labeled ISMs showed complete

0.8

d)

13C ISMs, DP6 showed complete degradation of monochloro-benzene in MNA

0.6

on (m

g/bd

WBC-2 13C MNA 13C k (per day) 0.019 0.021

half life (days) 37.1 33.5 and WBC-2

0.2

0.4

once

ntra

ti

66 % 69 %

( y )

0 Pre-Deployment WBC-2 13C MNA 13C

Co

20

4

-

9

PDB-01

PDB-04

PDB-07

PDB-10 (2009)

104

(200

(

Bio-Traps: 13C-labeled 2,500

3,000 13C Utilized for CO2, 13C Chlorobenzene

8 (NW) 104 (NW)

Chloro-benzene

1,000

1,500

2,000

C D

el (‰

)

(NW) 107 (NE) 6 (NE)

-500

0

500 DIC

Incorporation in dissolved

500 Background MNA Lactate/Chitin WBC-2

80 13C Utilized for Biomass, 13C Chlorobenzene

inorganic carbon = Mineralization

40

60

80

(‰)

PDB-01 PDB-04 PDB-07 PDB-10 9)

8 (NW) 104 (NW) 107 (NE) 6 (NE)

Incorporation in PLFA = Metabolism

20

0

20

PLF

A D

el )Metabolism

(C for growth)

-40

-20

Background MNA Lactate/Chitin WBC-2 21

,

uc ve

cy

QuantArray Microbial Analysis- Anaerobic

1 0E+06

1.0E+07

1.0E+08 -------Reductive dechlorination-------

1.0E+04

1.0E+05

1.0E+06

Cells

/mL

J < <

1.0E+01

1.0E+02

1.0E+03

Red ti dechlorination

DHC tceA vcr BVC DHBt DHG BCR bssA assA

MNA LAC WBC-2

Reductive dechlorination: DHC , Dehalococcoides spp. TCE, tceA reductase VCR, vinyl chloride reductase

BTEX, PAHs and alkanes: BCR, Benzoyl coenzyme A reductase bssA benzylsuc inate synthase BV1 , vinyl chloride reductase

DHBt, Dehalobacter spp. DHG, Dehalogenimonas spp.

bssA, benzylsuccinate synthase assA, alkylsuccinate synthase

22

1.0E 06

QuantArray Microbial Analysis- Aerobic

1.0E+06

1.0E+07

1.0E+08

1.0E+04

1.0E+05

Cells

/mL

< < <1.0E+02

1.0E+03

pMMO sMMO TCBO RDEG RMO PHE EDO PM1 ALKB

NA NA

pMMO, particulate methane monooxygenase

MNA LAC WBC-2

monooxygenase sMMO, soluble methane

monooxygenase TCBO, trichlorobenzene

PHE, phenol hydroxylase EDO, ethylbenzene/isopropyl-

benzene dioxygenase PM1 Methylibium petroliphilum

dioxygenase RDEG, toluene monooxygenase 2 RMO, toluene monooxygenase

PM1, Methylibium petroliphilum PM1

ALKB, alkane monooxygenase 23

t

Changing Paradigm Previous pp garadigm for chlorinated VOCs: • Anaerobic

reductive d hl i ldechlorinatiion only process in apparent low redox zones

•• Aerobic oxidation Aerobic oxidation requires measurable oxygen

• Anaerobic oxidation responsible for losses of lower VOCs at anaerobic plume fringesplume fringes

24

BioaugmentationBioaugmentation:: FracturedFractured

Inject Pump

FracturedFractured sedimentary rocksedimentary rock aquifer, formeraquifer, former

EOS & KB-1 qq

Naval Air WarfareNaval Air Warfare Center (NAWCCenter (NAWC))

“stall”?stall ?

25

Toxic Substances Hydrology Program New Jersey Water Science Center National Research Program

Matrix diffusion/Matrix diffusion/ DNAPL di l tiDNAPL di l tiDNAPL dissolutionDNAPL dissolution

Chl d I W ll

??

Chloride, Injection Well 36BR-A

Cl:excess Currently investigating

Cl-Bio

Currently investigating changes in native and bioaugmented microbial communities-

(Modified from Tom Imbrigiotta)

toxicity/inhibition effects cause growth of “partial dechlorinators”?

26

Use of laboratory testing to characterize microbialUse of laboratory testing to characterize microbial communities and biodegradation processescommunities and biodegradation processescommunities and biodegradation processescommunities and biodegradation processes

Native Bioreactor Bioaugmented Bioreactor

Polyethylene and polyurethane supportPolyethylene and polyurethane support matrix for building biofilm of native or bioaugmented microorganisms

27

4

DCB 12DCB

SCD Bioreactors- Aerobic vs. Anaerobic

8.0

C Reactor 8/13/12 aerobic

124TCB 8.0

D Reactor 8/10/12 anaerobic

124TCB

6.0

n (u

mol

/L) 123TCB

14DCB

13DCB

6.0

123TCB

14DCB

13DCB

12DCB

2.0

4.0

once

ntra

tion 12DCB

CB

B 2.0

4.0 CB

B

0.0 0 10 20 30 40

Co

Time (hrs)

0.0 0 10 20 30 40

Time (hrs)

• occurrence of aerobic and anaerobic degradation by native bacteria • aerobic degradation faster than anaerobic biodegradation

Time (hrs) Time (hrs)

• aerobic degradation faster than anaerobic biodegradation • WBC-2 able to degrade chlorinated benzenes and benzene anaerobically • accumulation of daughter products not evident

28

Questions?Questions?

29

Acknowledgements USGS MD-DE-DC Fate and Bioremediation Team Michelle Lorah

CRADA-Geosyntec Consultants Duane Graves

NRP Reston Collaborators Isabelle Cozzarelli Denise Akob

Michelle Lorah Jessica Teunis Mastin Mount Michael Brayton

Allen Shapiro Dan Goode Tom Imbrigiotta

Michael Brayton Charles Walker Roberto Cruz Emily Majcher

Claire Tiedeman

USEPA Region III Emily Majcher Anna Baker Luke Myers

US Army Aberdeen Proving Ground US Army, Aberdeen Proving Ground Installation Restoration Program Toxic Substances Hydrology

Program

30