Hanford 100H Cr(VI)

long- term Bioimmobilization

Hanford 100H Cr(VI)

long- term Bioimmobilization

Terry C. HazenDOE BER Distinguished Scientist

Head, Ecology DepartmentHead, Center for Environmental Biotechnology

Co-Director, Virtual Institute for Microbial Stress and SurvivalLawrence Berkeley National Laboratory

Berkeley, California 94720tchazen@lbl.gov http://vimss.lbl.gov

TimeTimeDistance from SourceDistance from Source

Dominant Terminal Electron Accepting ProcessDominant Terminal Electron Accepting Process

+10+10

00

-10-10

Electron AcceptorsElectron Acceptors

pEpE

AerobicRespiration

AerobicRespiration

O2 O2

OrganicsOrganics

O2 O2

SO4 SOSO44

SulfateReduction

SulfateReduction

SO4 SO4

H2 SHH22 SS

MethanogenesisMethanogenesis

CO2 CO2

CH4 CH4

H2 H2

DenitrificationDenitrification

NO3 NONO33

NO3 NONO33

Iron (III)ReductionIron (III)

Reduction

Fe (III)Fe (III)

Fe (II)Fe (II)Chemical SpeciesChemical Species

Equi

vale

nts

Equi

vale

nts

Critical BiogeochemistryCritical Biogeochemistry

PCE/TCE

Mn (IV)

Cr (VI)U (VI)

Fe(III) Fe(II)Microbial reduction

Fe(II) Cr(VI)+ Cr(III) Precipitation

Cr(VI) Bioreduction Lab Studies

+

Jiamin Wan, Tetsu Tokunaga, Mary Firestone, Eoin Brodie and Terry Hazen (ERSP/NABIR supported 1998-2004)

Tokunaga, T. K. J. Wan, M. K. Firestone, T. C. Hazen, K. R. Olson, D. J. Herman, S. R. Sutton, and A. Lanzirotti. 2003. In-situ reduction of Cr(VI) in heavily contaminated soils through organic carbon amendment. J. Environ. Qual. 32:1641-1649.

Tokunaga, T. K., J. Wan, T. C. Hazen, E. Schwartz, M. K. Firestone, S. R. Sutton, M. Newville, K. R. Olson, A. Lanzirotti, and W. Rao. 2003. Distribution of chromium contamination and microbial activity in soil aggregates. J. Environ. Qual. 32:541-549.

Tokunaga, T. K., J. Wan, M. K. Firestone, T. C. Hazen, E. Schwartz, S. R. Sutton, and M. Newville. 2001. Chromium diffusion and reduction in soil aggregates. Environmental Science & Technology 35:3169-3174.

.

0

0.2

0.4

0.6

0.8

1

frac

tio

n o

f ad

ded

Cr(

VI)

un

red

uce

d

0 100 200 300 400

days since OC addition

+0 ppm OCk = 7.0 E-8 s-1

+800 ppm OCk = 9.6 E-8 s-1

+4,000 ppm OCk = 2.0 E-7 s-1

+0 ppm OC+800 ppm OC+4,000 ppm OC

initially 1,000 ppm Cr(VI) in pore waters1 0 m m

1 0 m m

1 0 m m

1 ,4 0 0

1 ,2 0 0

1 ,0 0 0

8 0 0

6 0 0

4 0 0

2 0 0

0

+800 ppm OC

+80 ppm OC

+0 ppm OC

nativ

e

soil

[Cr]

day 31

100

10

1 mid

-dep

th

ou

ter

cen

ter

mid

-dep

th

ou

ter

cen

ter

mid

-dep

th

ou

ter

cen

ter

100

10

1

100

10

1

+800 ppm OC

+80 ppm OC

+0 ppm OC

dehy

drog

enas

e ac

tivity

, µg/

(g s

oil)

(7

days

incu

batio

n)

The Cr source is believed to be sodium dichromate (Na2 Cr2 O7 .2H2 O)

Hanford 100H

Cr Concentration MapCr Concentration Map Lithological ColumnLithological Column

Hanford 100H Site Characterization

Hanford 100D

Overall Objective

To carry out field investigations to assess the potential for immobilizing Cr(VI) in groundwater using lactate- stimulated bioreduction of Cr(VI) to Cr(III) at the Hanford 100H site, and to determine critical community structure changes and stressors that would enable control and predictions of fundamental biogeochemistry that enables this bioremediation strategy for Cr(VI)

Integrated ApproachField Measurements

Hydrogeology Geophysics Geochemistry and Isotopic Composition

Lab Measurements

Microbiology

Lactic Acid Molecule

H+ from water

OH- from water

HRC®

(Polylactate Ester)

HRC

Water samplers

Ringold clay

Injection of 40 lbs of 13C-labeled HRC Well 699-96-45, August 3, 2004

Pumping - 27 daysWell 699-96-44

Hanford sandy gravel and gravelly sand

Pumping

Groundwater level

Ringold silt

Injection at depths of 44 ft to 50 ft

Field HRC Injection Test

Post-HRC Injection Changes in Electrical Conductivity

High Ksat

HRC Injection Zone

2 Days after HRC injection

3 DAYS

30 DAYS

Groundwater Flow

Pump

Hypothesis: Lactic acid

Hypothesis: Reaction halo due to formation of precipitates

Lower Ksat

42’

45’

42’

45’

42’

45’

0.1

1

10

100

1000

7/5 7/12 7/19 7/26 8/2 8/9 8/16 8/23 8/30 9/6

Redox

DO

1.E+05

1.E+06

1.E+07

1.E+08

7/5 7/12 7/19 7/26 8/2 8/9 8/16 8/23 8/30 9/6

Bio

mas

s

Pumping waterWell 44 - pumpingWell 45 - injection well

HR

C in

ject

ion

and

pum

ping

beg

an

pum

p sh

ut o

ff

Max

imum

bi

omas

s

Results of HRC Biostimulation

6

6.5

7

7.5

8

8.5

9

9.5

10

7/5 7/12 7/19 7/26 8/2 8/9 8/16 8/23 8/30 9/6pH

Redox dropped from 240 to -130 mV

DO dropped from 9 mg/l (~100%) to 0.35 mg/l (4.5%)

D. vulgaris (direct fluorescent antibody)

1.E-04

1.E-03

1.E-02

1.E-01

1.E+00

1.E+01

1.E+02

8/2/04 9/1/0410/2/0411/1/0412/2/041/1/05 2/1/05 3/3/05 4/3/05 5/3/05 6/3/05 7/3/05 8/3/05 9/2/05

Fe(II

) Con

cent

ratio

n, m

g/L

44 pum4549.552.556.5Well 4

Iron Reduction

0

20

40

60

80

100

8/2 8/12 8/22 9/1 9/11 9/21

Nitr

ate

(ppm

)

0

20

40

60

80

100

120

140

160

180

200

8/2 8/12 8/22 9/1 9/11 9/21

Sulfa

te (p

pm)

Sulfate reductionSulfate reductionDenitrification Denitrification

Biogeochemical Evidence of Microbial Metabolism in Groundwater

Fe(III) Fe(II)Microbial reduction Fe(II) Cr(VI)+ Cr(III) Precipitation+

Average Soluble Cr(VI) Concentration

1.E-03

1.E-02

1.E-01

1.E+00

8/1/04 10/1/04 12/1/04 1/31/05 4/2/05 6/2/05 8/2/05

CrV

I con

cent

ratio

n (p

pm)

Injection well

Downgradient monitoring well

Upgradient monitoring well

d13C of Dissolved Inorganic Carbon is Byproduct of HRC Metabolism

Biogeochemical Evidence of Microbial Metabolism in Groundwater

-20

-10

0

10

20

30

40

50

60

70

80

Aug

-04

Oct

-04

Dec

-04

Feb-

05

Apr

-05

Jun-

05

Aug

-05

Oct

-05

Dec

-05

Jan-

06

Apr

-06

Jun-

06

Aug

-06

Oct

-06

Dec

-06

Jan-

07

Apr

-07

Jun-

07

δ13C

DIC

45 ft49.5 ft52.5 ft56.5 ft

δ13C of HRC

δ13C of background

Injection & Pumping Pumping Pumping

13C Phospholipid Analysis

General bacterial biomarkers indicate rapid enrichment in 13C13C ratio is greater than expected (overall spiked HRC ratio was 15 per mil)

13C polylactate used as spike it is not esterified to glycerol backboneit is released and consumed more rapidly

Biomarkers for Flavobacteriaceae increased following injection but showed minimal enrichment with 13C.

Flavobacteria do NOT typically utilize lactate, but may use glycerol (backbone, unlabeled)

DOE 16s rDNA microarray• Rapidly detect the

composition and diversity of microbes in an environmental sample

• Massive parallelism - 550,000 probes in a 1.28 cm2

array• all 9,900 species in 16S

rDNA database• Single nucleotide mismatch

resolution

MATCHMISMATCH

cctagcatgCattctgcatacctagcatgGattctgcata

Days since HRC injection0 11 17 27

Cor

rect

ed h

ybrid

izat

ion

inte

nsity

0

500

1000

1500

2000

2500

3000 Desulfovibrio halophilus Geobacter metallireducens Dechloromonas agitatus Pseudomonas putida

Microarray analysis of bacterial community changesduring Cr(VI) remediation at Hanford 100H site:

Dynamics of some significant organisms.

Injection Day

30 days later

Data mining – Bidirectional clustering

I I I I I MMMMMI MM I I MMMMI I I I I I I I MMMMMMM

Acetoclastic & Hydrogenotrophic Methanogenic Archaea

Sulfate reducers, Iron reducers

Nitrate reducers, Sulfate reducers

Crenarchaeotes

312 & 216 daysLower depths

I & M

Pre-injection9 & 112 daysMost depths

I & M17 days

Most depthsI & M

312 & 216 Upper depths

Nitrate reducers, sulfur oxidizers

Fermenters, Nitrate reducers, Sulfate reducers

9 & 17 daysUpper depths

I

9 daysAll depths

M 216 daysMost depths

M

I = Injection wellM = Monitoring well

Highest intensityLowest intensity

Functional groups – Iron reduction

Days post HRC injection Days post HRC injection

Hyb

ridiz

atio

n in

tens

ity

Fe(II) can abiotically reduce Cr(VI) to Cr(III)

Functional groups – Sulfate reduction

Days post HRC injection Days post HRC injection

Hyb

ridiz

atio

n in

tens

ity

H2 S can abiotically reduce Cr(VI) to Cr(III)

Functional groups – Methanogenesis

Days post HRC injection Days post HRC injection

Hyb

ridiz

atio

n in

tens

ity

Presence of methanogens indicates strongly reducing conditions

Functional microarray analysis

0

100

200

300

400

500

600

700

800

900

45ft 49.5ft 52.5ft 56.5ft 45ft 49.5ft 52.5ft 56.5ft

Chromium

Nitrate

Cytochrome

Sulphate

Methanogenesis

Methane oxidation

Injection well Monitoring well

Nitrate, Sulfate, Iron reduction. Methanogenesis, Methane oxidation, Sulfur oxidation.Many chromium tolerance/reduction genes.

Mea

n si

gnal

to n

oise

ratio

Joe Zhou, Joy Van Nostrand - Oklahoma

April 17th 2006

Affymetrix PhyloChip Fluorescence

Scan

System-specific NimbleGen chip

+ NanoSIMS13C:12C analysis

rRNA profile showsspecies that are present

in the community

Indicates subset of active community that consumed

13C-label substrate

NanoSIMS + microarray indicates active organisms

----100 μm----

12C rRNA13C rRNA

Array spotted with universal 16S

probe set

“Chip-SIP” yields identity and function from the same sample

(Hoeprich, Pett-Ridge, Brodie, et al. New Genomics:GTL project)

DNA

RNA

Protein

Population

Community

Ecosystem

Cell

EcologyComputational

EcologyGeochemistryComputational

Desulfovibrio are present at elevated numbers during biostimulation

How do cellular responses to relevant field conditions impact cellular activities and survival?

Systems Biology to Elucidate Field Relevant Responses

Desulfovibrio spp. have been observed as predominant populations at Hanford 100-H during biostimulation (Hazen et al.)

GenomicProteomicMetabolomicComputational

What is Known from Hanford 100H Lactate Biostimulation Experiment

• In situ hydrogeological, geochemical (including radioactive and stable isotope analyses), geophysical measurements, and microbiological analyses of water samples and sediments provided detailed and robust interpretation of field-scale biogeochemical processes.

• The 13C concentration, which was used to label the injected lactate, is essential to track and identify the presence of microbial metabolism and lactate degradation.

• A sequential depletion of competing terminal electron acceptors O2-, NO3-, Fe3-,SO4

2-,

and transiently CO2 , creating a sustained dissolved, ferrous ion environment.

• A chemical reaction of ferrous ion with toxic and soluble Cr(VI) causes the formation of nontoxic and insoluble Cr(III)-Fe complexes.

• Cr(VI) concentration has remained below the drinking water standards for ~3 years after a single 40lb polylactate injection.

• The longevity of the Cr(VI) bioimmobilization indicates an efficacy of using lactate injection for controlling the Cr(VI) concentration in groundwater at many contaminated sites.

What Cellular Systems are Involved in Cr(VI) Responses in Desulfovibrio vulgaris Hildenborough?

• Sulfate influx down- expressed

• Metal efflux up-expressed

• chrAB up-expressed

• FMN dependent nitroreductase, NADH dehydrogenase, and FMN reductase up-expressed

Klonowska, A., He*, Z., He, Q., Hazen*, T.C., Thieman, S.B., Alm*, E.J., Arkin*, A.P., Wall*,J.D., Zhou*, J. and Fields*, M.W. Global Transcriptomic Analysis of Chromium(VI) Exposure of Desulfovibrio vulgaris Hildenborough Under Sulfate-Reducing Conditions. (in press)

FMN red.NADP+ + FMNH2

NADPH2 + FMN NADP dehy

nitroreductase

Cr(VI)

Cr(III)

ChrB

ChrA

Metal ATPase

MerP AcrA OM Efflux

Drug Resist.

PerRZur

Fur

SO4Influx

Cr(III)

Cr(III)

Cr(III)

Cr(

III)

Cr(

III) C

r(III)

SO4Influx

SO4

Influ

x

-DNA repair-Protein repair

AcknowledgmentsAdam Arkin, Eric Alm, Kat Huang, Dylan Chivian, Janet Jacobson, Adam Arkin, Eric Alm, Kat Huang, Dylan Chivian, Janet Jacobson, Jay Keasling, Jay Keasling, Aindrila Aindrila Mukhopadhyay, Eoin Brodie, Sharon Borglin, HoiEoin Brodie, Sharon Borglin, Hoi--Ying Holman, Jil Geller, Ying Holman, Jil Geller, Elenor Woezi, Jenny Lin, Dominique Joyner, Rick Huang, Romy ChakElenor Woezi, Jenny Lin, Dominique Joyner, Rick Huang, Romy Chakraborty, raborty, Boris Faybishenko, Mark Conrad, Joern Larsen, Zouping Zheng, GarBoris Faybishenko, Mark Conrad, Joern Larsen, Zouping Zheng, Gary Andersen, y Andersen, Todd DeSantis, Tetsu Tokunaga, Jiamin Wan, Susan Hubbard, Ken WiTodd DeSantis, Tetsu Tokunaga, Jiamin Wan, Susan Hubbard, Ken Williams, lliams, John Peterson, Natalie Katz, Jill Banfield, Tamas Torok, John Peterson, Natalie Katz, Jill Banfield, Tamas Torok, Seung Baek, Don Seung Baek, Don Herman, Mary FirestoneHerman, Mary Firestone

Stephen Sutton, Matthew NewvilleStephen Sutton, Matthew Newville

Paul Richardson, Paul Richardson, Phil HugenholtzPhil Hugenholtz

Phil Long, et al.Phil Long, et al. Steve Koenigsberg, Ana WilletSteve Koenigsberg, Ana Willet

U Washington

Mathew Fields, et. al.Mathew Fields, et. al.

Jizhong Zhou, et. al.Jizhong Zhou, et. al.

David Stahl, et. al.David Stahl, et. al.

Judy Wall, et. al.Judy Wall, et. al.

Anup Singh, et. al.Anup Singh, et. al.

Martin Keller, et. al.Martin Keller, et. al.

Eric Alm