evaluating the efficacy of bioremediation of …evaluating the efficacy of bioremediation of uranium...

Evaluating the Efficacy of Evaluating the Efficacy of Bioremediation of Uranium Bioremediation of Uranium in the Subsurfacein the Subsurface

Philip E. Long

Pacific Northwest National Laboratory

Federal Remediation Technologies Roundtable15 November 2007 EPA Conference Center, Arlington, VA

2

Outline of presentationOutline of presentationOutline of presentation

Background: persistence of uranium groundwater plumesConcept for uranium bioremediationApproach to development of a mechanistic understanding of subsurface mobility of uranium at the field scaleEvaluation of uranium bioremediationFuture directions and developments

3

Persistence of uranium groundwater plumes (U(VI) Concentrations (mg/L))

Persistence of uranium groundwater plumesPersistence of uranium groundwater plumes (U(VI) Concentrations (mg/L))(U(VI) Concentrations (mg/L))

Presenter�

Presentation Notes�

At the Old Rifle site, uranium (~1-2uM) is the risk driver for the site and is present at low but significant concentrations across the site, particularly in the subsurface beneath where the spent ore tailings were piled. Our test site is located within the area of high(er) concentrations of uranium in the groundwater. The subsurface is dominated by braided deposits of sand and coarse gravels and cobbles. Depth to the confining layer is approximately 20 ft. Saturated aquifer thickness is about 12ft. The site is naturally anoxic with abundant Fe(II) in the sediments, occasional spikes in nitrate concentration and low DO near the water table. The current remediation decision for the site is natural flushing. �

4

Well 655

Persistence of uranium groundwater plumes (U(VI) Concentrations (mg/L))

Persistence of uranium groundwater plumesPersistence of uranium groundwater plumes (U(VI) Concentrations (mg/L))(U(VI) Concentrations (mg/L))

5

Concept for U(VI) bioreductionConcept for U(VI) Concept for U(VI) bioreductionbioreduction

U(VI) is the mobile valence state of uraniumReduced uranium, U(IV), is insoluble as uraniniteReduction of U(VI) to U(IV) within aquifers could precipitate and immobilize uraniumLab studies suggest simple strategy to promote U(VI) reduction in contaminated aquifers:

add acetate as an electron donor to stimulate dissimilatory metal-reducing microorganismsU(VI) is reduced concurrently with Fe(III)

C CC C

Fe(II)

CO2

e-

Fe(III)

U(VI)

U(IV)U(IV)

C CH3

O−ONa+

Acetate structureLactate structure

• Typical human hair diameter:100,000 nanometers (nm)• Geobacter cell at left 300 nm wide• “Nanowires” ~10 nm(image from Reguera et al. Nature Vol 435, 23 June 2005)

7

Implementation of in situ bioremediation of U(VI)

Implementation of Implementation of in situin situ bioremediation bioremediation of U(VI)of U(VI)

Acetate Solution(Low Conc.)DO < 1mg/L

N2

InjectionGallery

Pump or Gravity feed

Acetate

GroundwaterFlow

CH3COO-

CH3COO-

CH3COO-

CH3COO-

CH3COO-

CO2

Fe(III)Fe(II)

CH3COO-

U(VI)U(IV)

U(VI)U(VI)

U(VI)

U(VI)U(VI)

U(VI)

U(VI)

U(VI) U(VI)

U(VI)

U(VI)

Zone of U(VI) Removal}

8

Context for metal and radionuclide bioremediation

Context for metal and radionuclide Context for metal and radionuclide bioremediationbioremediation

Pump and treatForced gradient

amendment

Natural gradient

displacive amendment

Natural gradient non-

displacive amendment

Monitored natural

attenuation

Usually the most costly,

may be required for hydraulic control

Cost for maintaining

gradient. Control on flow

direction

Difficult to separate injectate

dilution from microbial effects

Minimal dilution effect. Limited donor concentration

Contaminants may not

respond in desired time

frame

Active Passive

Remed

ial

appro

ach

~Decreasing cost

Commen

ts

Bioremediation

9

Context for field bioremediation research at the Old Rifle Uranium Mill Tailings Site Context for field bioremediation research Context for field bioremediation research at the Old Rifle Uranium Mill Tailings Siteat the Old Rifle Uranium Mill Tailings Site

Presenter�


The Old Rifle Site is one of these uranium ore extraction facilities. The site is located in the town of Rifle just off of I-70 on the Western slope of Colorado. The tailings pile, extraction facilities, ore storage areas, basically most surface structures and stored materials have since been removed from the site in accordance with Uranium Mill Tailings Remedial Action program. The top 4 to 7 feet of surface soil was also removed and a clay cap put into place prior to re-soiling and planting. Our test plot is located within an area of site where the spent tailings were stored.�

12

Conceptualized Test Plot

Observation wellsControl Wells

Injection Gallery Detail

Acetate

GW flow

25 ft

50 ft

80 ft

Injection Gallery

1ft

5ft

B-01

0.00.20.40.60.81.01.21.4

B-02

0.00.20.40.60.81.01.21.4

B-03

Days

0 20 40 60 80 1000.00.20.40.60.81.01.21.4

M-01

0.00.20.40.60.81.01.21.4

M-02

0.00.20.40.60.81.01.21.4

M-03

0.00.20.40.60.81.01.21.4

M-04

0.00.20.40.60.81.01.21.4

M-05

0 20 40 60 80 1000.00.20.40.60.81.01.21.4

M-06

0.00.20.40.60.81.01.21.4

M-07

0.00.20.40.60.81.01.21.4

M-08

0.00.20.40.60.81.01.21.4

M-09

0.00.20.40.60.81.01.21.4

M-10

0 20 40 60 80 1000.00.20.40.60.81.01.21.4

M-11

0.00.20.40.60.81.01.21.4

M-12

0.00.20.40.60.81.01.21.4

M-13

0.00.20.40.60.81.01.21.4

M-14

0.00.20.40.60.81.01.21.4

M-15

0 20 40 60 80 1000.00.20.40.60.81.01.21.4

U(VI) (uM)

Previous work at the Rifle SitePrevious work at the Rifle SitePrevious work at the Rifle Site

CFBfirmicutesbetaactinobacteriaSpirochetesDesulfosporosinusotherVerrumicrobiaother deltaSO4 Red - deltaGeobacteraceae

18 24 39

Days Since Beginning Acetate Injection%

16S

rDN

Ase

quen

ces

240%

20%

40%

60%

80%

100%

Background M-07

Presenter�


Research at the site has focused on stimulating the in situ activity of metal-reducing microorganisms in the subsurface to reduce and immobilize uranium from the groundwater at this site. This has been accomplished through the installation of injection galleries upgradient of a series of monitoring wells. Acetate is injected into the aquifer over time and changes in the subsurface are monitored via groundwater and sediment sampling in and among the downgradient monitoring wells. In all tests geochemical data has been collected in concert with advanced molecular biology techniques to link changes in geochemistry with changes in microbial community composition. This approach will be emphasized even further under the new IFC and will also be linked with geophysical changes detected in the subsurface. �

13

U(VI) Loss at 6 meters from B-02 to M-08

-100-80-60-40-20

020406080

100

11/5/01 5/24/02 12/10/02 6/28/03 1/14/04 8/1/04

Date

-1.5

-1

-0.5

0

0.5

1

1.5

2

U(V

I) (u

M) l

oss % U(VI) lost

2002 Acetate Start2002 Acetate End2003 Acetate Start2003 Acetate EndU(VI) (uM) lost

U(VI) loss for both 2002 and 2003 experiments

U(VI) loss for both 2002 and 2003 U(VI) loss for both 2002 and 2003 experimentsexperiments

14

0

10

20

30

40

50

60

70

80

90

100

May 20

02Ju

ne 20

02Ju

ly 20

02

Augus

t 200

2

Septem

ber 2

002

Octobe

r 200

2

Novem

ber 2

002

Decem

ber 2

002

June

2003

July

2003

Augus

t 200

3

Septem

ber 2

003

Octobe

r 200

3

Novem

ber 2

003

Janu

ary 20

04

Februa

ry 20

04Marc

h 200

4Apri

l 200

4May

2004

June

2004

July

2004

Novem

ber 2

004

Februa

ry 20

05Apri

l 200

5Ju

ne 20

05

Decem

ber 2

005

Septem

ber 2

006

Decem

ber 2

006

% U

(VI)

Rem

aini

ng

2002 Acetate

Amendment

2003 Acetate

Amendment

The Unexpected Continued Removal of U(VI)

U(VI) loss between B02 and M08

The Unexpected Continued Removal of U(VI)The Unexpected Continued Removal of U(VI)

U(VI) U(VI) loss between B02 and M08loss between B02 and M08

Source: N’Guessan et al. 2007 ES&T in review

15

Firmicutes predominate in post-amendment reduced sediment

Distribution of 16S rRNA gene sequences

FirmicutesFirmicutes predominate in postpredominate in post--amendmentamendment reduced sedimentreduced sediment

Distribution of 16S Distribution of 16S rRNArRNA gene sequencesgene sequences

Source: N’Guessan et al. 2007 ES&T in review

16

Passive multilevel samplers. A. Cell on support rod being lowered into monitoring well. B. MLS cells from a background well. C. MLS cells from a treatment zone well undergoing sulfate reduction.

A. B.

C.

Rifle Integrated Field Challenge Site:Rifle Integrated Field Challenge Site:Rifle Integrated Field Challenge Site:Objective: development of a mechanistic understanding of subsurface mobility of uranium at the field scaleTesting hypotheses relating to:

Extension of Fe-reducing conditionsU(VI) Sorption under reducing conditionsMechanisms for post-biostimulation U removalRates of natural bioreduction of U

Key approaches:Mechanisms of U bioreduction illuminated by protein expressionRelative contribution of biotic processes and abiotic uranium immobilization processes evaluated (e.g. U bioreduction and U sorption)Correlation of subsurface geochemical processes with geophysical monitoring of subsurface redox status associated with bioreductionComprehensive reactive transport modeling of uranium mobility in the subsurface

Presenter�


The overall science themes for the Old Rifle IFC build on and extend earlier research observations and incorporate new techniques into the research plan. For example: we will attempt to deploy proteomics techniques to monitor stimulated subsurface microbial communities during metal reduction in a testable format. These data will be closely coupled with gene expression, genomic and community sequencing efforts to provide the most cutting edge description of microbial community dynamics yet attempted in subsurface environments. We will evaluate the major immobilization processes for uranium in detail to assess the relative importance of enzymatic reduction, sorption, mineralization and the potential, if any, for abiotic uranium reduction. We will examine stimulated changes in subsurface processes (TEAPs) and correlate these observations with geophysical measurements taken at the surface in order to evaluated new noninvasive ways of monitoring uranium bioremediation from the surface. Also, we will incorporate the collected data into a comprehensive reactive transport model for the site that should provide not only a better accounting of the major factors affecting uranium mobility at this UMTRA site but also a simulation of in situ metal reduction and uranium bioremediation. �

18

2007 field experimentReplicated earlier field experiments showing U(VI) bioreductionby GeobacterIntentionally limited acetate during part of the experimentSuccessfully generated samples for proteomic and metagenomic analysis Data sets include: U(VI) removal rates, hydraulic conductivity, hydrogeophysical monitoring, gene expression data, mineralogical changes, in situ incubators/sensorsDirect access to naturally bioreduced sediment (and uranium?)

19

Winchester Well Layout and Distribution of Reduced Sediments

Winchester Well Layout and Distribution of Winchester Well Layout and Distribution of Reduced SedimentsReduced Sediments

20

Fe(II) and U(VI) trendsFe(II) and U(VI) trendsFe(II) and U(VI) trends

D-01

0

1

2

3

4

5

6

7

19-Jul-07 29-Jul-07 8-Aug-07 18-Aug-07 28-Aug-07 7-Sep-07 17-Sep-07 27-Sep-07

Fe(I

I) (

mg

/l)

0

50

100

150

200

Ace

-/B

rom

ide (

mM

)U

(VI)

ug/l

MCL

U(VI) Fe(II)

Injection InjectionGW flush

21

Uranium Concentration (μM) as a function of time (days)Uranium Concentration (Uranium Concentration (μμM) as a function of time (days)M) as a function of time (days)

22

Proteomic Sampling and Characterization of the Microbial Community Structure and Dynamics During Electron Donor Amendment at the Rifle IFC

• In groundwater from downgradient wells, proteins from six different Geobacter species were detected where G. bemidjiensis was the most abundant organism•Protoemic data are also being used to characterize the microbial community from stimulated sediments•Proteomic data are being obtained both at EMSL/PNNL and ORNL, and analyzed jointly with UC Berkeley

High throughput 9.4 Tesla FTICR(PNNL/EMSL)

FT-MS Orbitrap(ORNL)

Tangential Flow Filtration (TFF) U Mass

G. m

etal

lired

ucen

s

s i s 2 s s

S a m p l e s e t 1 ( 8 / 1 7 D - 0 ? )S a m p l e s e t 2 ( 8 / 2 3 D - 0 5 )

G. b

emid

gien

sis

G. l

ovle

yi

G. s

p_FR

C_3

2

G. s

ulfu

rred

ucen

s

G. u

rani

umre

duce

ns

Pro

tein

s

0

100

200

300Well D-07 (8/17/07)Well D-05 (8/23/07)

Presenter�


SBY: At some point, you have to make the case for doing the proteomics – you might focus on the uniqueness of site-specific conditions with regard to one-size does not fit all �

23

Evaluation of uranium bioremediationEvaluation of uranium bioremediationEvaluation of uranium bioremediation

CharacterizationConceptual model developmentMonitoringNumerical modeling

24

CharacterizationCharacterizationCharacterization

Importance of Sediment Grain Size to U sorptionImportance of Sediment Grain Size to U sorptionImportance of Sediment Grain Size to U sorption

Uranium 0.5 M Nitric Acid Extractions for "D-08 16"

0.000

2.000

4.000

6.000

8.000

10.000

12.000

14.000

16.000

18.000

20.000

< 53

micr

on

<106

> 53

micr

on

< 149

> 1

06 m

icron

< 250

> 1

49 m

icron

< 500

> 2

50 m

icron

< 100

0 > 5

00 m

icron

<2000

>10

00 m

icron

<2000

micr

on

Grain Size Fraction

U c

onc e

ntra

tion

(μg/

gse

d im

ent)

25

MonitoringMonitoringMonitoring

Seasonal changesAppropriate spatial coverageReal time monitoringEvent-based samplingPassive in situ geochemical and biological sampling

26

2002 2003 2004 2005 2006

Water Table Elevation, Rifle Site 2001 to 2006

27

Stratified Water ChemistryStratified Water ChemistryStratified Water Chemistry

Depth-dependent U(VI) and DOHighest DO and U(VI) near the water tableIssues

Oxygen diffusion through water tableBackground utilization of DOScreened interval of wells

Background MLS U(IV) and DO

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

5/24/02 9/1/02 12/10/02 3/20/03 6/28/03 10/6/03 1/14/04

Date

B-01 Water table elevation

1614.6

1614.8

1615

1615.2

1615.4

1615.6

1615.8

1616

1616.2

1616.4

Ele

vati

on

(m

)

2002 Acetate addition 2003 Acetate addition

Ele

vatio

n (m

)D

O (m

g/l)

and

U(V

I ) ( μ

M)

Biogeochemical Reaction NetworkBiogeochemical Reaction NetworkBiogeochemical Reaction NetworkMultisite, multicomponent uranium surface complexation model

SSOH + UO22+ = SSOUO2

+ + H+ Log K = 6.798SOH + UO2

2+ = SOUO2+ + H+ Log K = 5.817

WOH + UO22+ = WOUO2

+ + H+ Log K = 2.57SSOH + UO2

2+ + H2O = SSOUOOH + 2H+ Log K = -0.671SOH + UO2

2+ + H2O = SOUOOH + 2H+ Log K = -2.082WOH + UO2

2+ + H2O = WOUOOH + 2H+ Log K = -5.31823 uranium aqueous complexation reactions91 abiotic species, minerals, surface sites3 energetics-based acetate-oxidizing TEAP reactions with biomass yieldDual Monod Rate Law with thermodynamic constraints

Iron TEAP0.125CH3 COO- + 0.6FeOOH(s) + 1.155H+ + 0.02NH4

+ = 0.02BM_iron + 0.6Fe++ + 0.96H2 O + 0.15HCO3-

Sulfate TEAP0.125CH3 COO- + 0.0057H+ + 0.0038NH4

+ + 0.1155SO4-- = 0.0038BM_sulfate + 0.0114H2 O + 0.231HCO3

- + 0.1155HS-

Uranium TEAP0.1250CH3 COO- + 0.3538H2 O + 0.0113NH4

+ + 0.3875UO2++ = 0.0113BM_iron + 0.855H+ + 0.1938HCO3

- + 0.3875UO2 (s)

⎟⎟⎠

⎞⎜⎜⎝

⎛⎟⎟⎠

⎞⎜⎜⎝

⎛Δ−⎟⎟

⎠

⎞⎜⎜⎝

⎛+Δ−

+=

−+

−+

RTGCOOCHH

HCOFeRTGkR r /}{}{

}{}{log3.2exp1]Ac[K

[Ac]sites] surface free[ min3

15

23

820

Ac

⎟⎟

⎠

⎞

⎜⎜⎝

⎛⎟⎟⎠

⎞⎜⎜⎝

⎛Δ−⎟⎟

⎠

⎞⎜⎜⎝

⎛+Δ−

++=

−

−

−

−−

− RTGCOOCHSO

HCOHSRTGSOK

SOkR rso

/}}{{

}}{{log3.2exp1]Ac[K

[Ac]][

][min

32

4

230

Ac2

44

42

⎟⎟

⎠

⎞

⎜⎜

⎝

⎛⎟⎟⎠

⎞⎜⎜⎝

⎛Δ−⎟⎟

⎠

⎞⎜⎜⎝

⎛+Δ−

++=

−+

−+

RTGCOOCHUO

HCOHRTGaqVIUK

aqVIUkR rU

/}{}{

}{}{log3.2exp1]Ac[K

[Ac]])([

])([min

342

2

23

90

Ac

30

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 25 50 75 100 125 150 175Time (d)

U(VI

) (uM

)

M-01M-02M-03M-04M-05Model

Biostimulation123 Days

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 25 50 75 100 125 150 175Time (d)

U(VI

) (uM

)



0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 25 50 75 100 125 150 175Time (d)

U(VI

) (uM

)



Reactive Transport Modeling of Uranium Bioreduction

Reactive Transport Modeling of Uranium Reactive Transport Modeling of Uranium BioreductionBioreduction

Initial aqueous U(VI) spatially variable Initial timing of aqueous U(VI) removal reproduced by model Uranium rebound is slower than model prediction Considerations

Sulfate reducers removing some uranium Uranium adsorption retarding front

Row 1

Row 2

Row 3

Exploring effects of physical and chemical heterogeneities on spatial patterns of calcite precipitation

Homo conductivity Homo solid Fe field

Hetero conductivity Homo solid Fe field

All three cases: same average flow velocity, and same total solid iron contentHeterogeneous conductivity field obtained from inverse modeling of tracer dataHeterogeneous Fe content: negative correlation with conductivity

Hetero conductivity Hetero solid Fe field

Physical and chemical heterogeneities lead to locally larger amounts of calcite precipitation, therefore increases the possibility of clogging.

Carl Steefel, Li Li (LBNL)

32

Future directions and developmentsFuture directions and developmentsFuture directions and developments

In-field monitoring of selected genes and proteinsCoupling of reactive transport models with in silico microbial models

33

Field Portable Microarray Analysis of Groundwater and Sediment

Field Portable Field Portable MicroarrayMicroarray Analysis of Analysis of Groundwater and SedimentGroundwater and Sediment

ERSP ScienceERSP Science

• Breadboard lab instrument

• Automated methods for direct detection of RNA

• Environmentally relevant samples, biomass and sample sizes

• Environmentally relevant and vetted microarray probes

• Statistical techniques for converting noisy data into actionable information

Rifle IFC Science/Rifle IFC Science/Generation 1 ProductsGeneration 1 Products

Rifle IFC/DOE SBIR Rifle IFC/DOE SBIR Generation 2 ProductsGeneration 2 Products

• End-to-end kits

• Large-volume sample preparation device and chemistry

• Amplification cocktails and reagents

• Field portable analysis instrument

• Semi-automated algorithms and software

Sample-to-answer integrated cartridges

Portable controller instrument = fluidics, microarray analysis, automated reporting. Insert cartridge and press “go”

Darrell P. Chandler, Chief Science OfficerAkonni Biosystems

34

Field-Portable Kit Process flow

FieldField--Portable KitPortable Kit Process flowProcess flow

Collect and concentrate sampleGroundwater or sediment

Bead-beater lysis (5 min)Universal, flow-through nucleic acid purification and concentration preparation (15 min)

Will isolate both DNA and RNA simultaneouslySimultaneous asymmetric amplification + microarray hybridization in same flow cell (120 min)

Single-pot DNA or RNA amplification and labelingWash (5 min)Imaging, data extraction, analysis and reporting (5 min)= 2.5 hours, sample-to-answer microbial community profiling

Rifle site

Sample Prep

Array

Community Profiles

Responsive organisms

+

35

Constraint-Based In Silico ModelingConstraintConstraint--Based Based In In SilicoSilico ModelingModeling

Genetic characterization of reaction pathwaysLaboratory characterization of flux constraintsOptimization under specific conditions

Biological Information

Understand Metabolism

In silico Cellular Models

Predict Growth Physiology

Analyze high-throughput data

Slide material from R. Mahadevan (Univ. of Toronto)

36

Conclusions on U(VI) bioremediation (natural gradient, non-displacive)

Conclusions on U(VI) bioremediation Conclusions on U(VI) bioremediation (natural gradient, non(natural gradient, non--displacivedisplacive))

Effectively removes U(VI) from groundwaterAdditional field-scale research needed understand mechanisms and durabilityPrecise monitoring of microbial activity on the horizonElectron donor pulsing and “engineering” of specific precipitates in situ may enhance long-term stabilityCompatible with monitored natural attenuationAmenable to regulatory evaluation

AcknowledgementsAcknowledgementsAcknowledgementsFunding for documenting the prerequisite characterization, conceptual model requirements, and monitoring requirements is provided by the U.S. Nuclear Regulatory CommissionFunding for all three IFC’s (Rifle, CO, Hanford 300 Area, and ORNL) is provided by the U.S. Department of Energy, Office of Science, Biological and Environmental Research, Environmental Remediation Sciences DivisionPacific Northwest National Laboratory is operated by Battelle Memorial Institute for the United States Department Of Energy under Contract DE-AC06-76RL01830

38

Rifle IFC Project ParticipantsRifle IFC Project ParticipantsRifle IFC Project ParticipantsJ. Banfield2, R. Bush3, K. Campbell4, D.P. Chandler5, J.A. Davis4, R. Dayvault6, J. Druhan2, H. Elifantz7, A. Englert8, R. L. Hettich9, D. Holmes7, S. Hubbard8, J. Icenhower1, P.R. Jaffe10, L.J. Kerkhof11, R.K. Kukkadapu1, E. Lesher12, M. Lipton1, D. Lovley7, S. Morris6, S. Morrison6, P. Mouser7, D. Newcomer1, L. N’Guessan7, A. Peacock13, N. Qafoku1, C. T. Resch1, F. Spane1, B. Spalding9, C. Steefel8, N. VerBerkmoes9, M. Wilkins2, K.H. Williams8, S.B. Yabusaki1

1. Pacific Northwest National Laboratory, Richland, WA 988162. University of California, Berkeley; Berkeley, CA 947203. Legacy Management, U.S. Department of Energy, Grand Junction, CO 815034. U.S. Geological Survey, Menlo Park, CA 940255. Akonni Biosystems, Frederick, Maryland 21701 6. SM Stoller, Inc., Grand Junction, CO 815037. Department of Microbiology, University of Massachusetts, Amherst, MA 010038. Lawrence Berkeley National Laboratory, Berkeley, CA 947209. Oak Ridge National Laboratory, Oak Ridge, TN 3783010.Dept. of Civil and Environmental Engineering, Princeton University, Princeton, NJ

0854411.Institute of Marine and Coastal Sciences, Rutgers University, Brunswick, NJ 0890112.Division of Environmental Science and Engineering Colorado School of Mines,

Golden, CO 8040113.Microbial Insights, Inc., Rockford, TN 37853

evaluating the efficacy of bioremediation of …evaluating the efficacy of bioremediation of uranium...

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