Earth Sciences Division, Ecology Department Field Investigations of Lactate-Stimulated Bioreduction...

Earth Sciences Division, Ecology DepartmentEarth Sciences Division, Ecology Department

Field Investigations of Lactate-Field Investigations of Lactate-Stimulated Bioreduction of Cr(VI) to Stimulated Bioreduction of Cr(VI) to

Cr(III) Cr(III) at Hanford 100Hat Hanford 100H

Field Investigations of Lactate-Field Investigations of Lactate-Stimulated Bioreduction of Cr(VI) to Stimulated Bioreduction of Cr(VI) to

Cr(III) Cr(III) at Hanford 100Hat Hanford 100H

Terry C. HazenTerry C. Hazen, B. Faybishenko, E. Brodie, D. Joyner, S. E.

Borglin, R. Chakraborty, M. Conrad, T. Tokunaga, J. Wan, S.

Hubbard, K. Williams, J. Peterson, M. Firestone, G.

Andersen, T. DeSantis, P. E. Long, D. R. Newcomer, A.

Willett, and S. Koenigsberg

LBNL, UCB, PNNL, Regenesis


Fe(III) Fe(II)Microbial reduction

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

Mesoscale Studies on Cr(VI) Bioreduction that led to Field Studies

+

Jiamin Wan, Tetsu Tokunaga, Mary Firestone and Terry Hazen (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

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 waters

0

0.2

0.4

0.6

0.8

1

0 100 200 300 400

days since OC addition

+0 ppm OCk = 1.5 E-8 s-1

+800 ppm OCk = 2.1 E-8 s-1

+4,000 ppm OCk = 4.4 E-8 s-1

initially 10,000 ppm Cr(VI) in pore waters

.

10 mm

10 mm

10 mm

1,400

1,200

1,000

800

600

400

200

0

+800 ppm OC

+80 ppm OC

+0 ppm OC

day 31

. .

100

10

1

100

10

1

100

10

1

+800 ppm OC

+80 ppm OC

+0 ppm OC


Multidisciplinary TeamMultidisciplinary TeamScientific Field LBNL PNNL Regenesis

Microbiology Terry Hazen,Eoin Brodie, Sharon Borglin, Dominique Joyner, Mary Firestone

Hydrogeology Boris Faybishenko, Jiamin Wan, Tetsu Tokunaga

Philip E. Long, Bruce Bjornstad

Geophysics Susan Hubbard, Ken Williams, John Peterson,

Geochemistry Mark Conrad Tom Resch, Kirk Cantrell

Field and technical support

Victor Gruol, Phil Rizzo Darrell Newcomer Steve Koenigsberg, Anna Willet, Kevin Lapus


Overall Objective 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 Approach

Field Measurements

Hydrogeology Geophysics Geochemistry and Isotopic Composition

Lab Measurements

Microbiology


The Cr source is believed to be sodium dichromate (Na2Cr207.2H2O)

Hanford 100H

Cr Concentration MapCr Concentration Map Lithological ColumnLithological ColumnHanford 100H Site CharacterizationHanford 100H Site Characterization

Hanford 100D

http://esd.lbl.gov/ERT/hanford100h/


LiBr Injection (2/27/2004)

LiBr Injection: 15.3 g/liter, 12 liters over 2 hours

55 Days No pumping 63 Days Pumping

63 Days Pumping + 2nd Injection

Lactic Acid Molecule

H+ from water

OH- from water

HRC®

(Polylactate Ester)


Lactate-Induced Bioreduction of Cr(IV)


HRC

Water samplers

Ringold clay

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

Pumping - 27 days Well 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 TestField 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

Biomass

Pumping waterWell 44 - pumpingWell 45 - injection well

HRC injection and pumping began

pump shut offMaximum biomass

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 9/6

pH

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)


Dissolved Oxygen (mg/l)

0

1

2

3

4

5

6

7

8

9

10

8/1/04 10/31/04 1/30/05 5/1/05 7/31/05 10/30/05

HRC injectionPumping

Injection well

Downgradient monitoring well

Upgradient monitoring well

Redox Potential (mV)

-200

-100

0

100

200

300

400

8/1/04 10/31/04 1/30/05 5/1/05 7/31/05 10/30/05


pH

5

6

7

8

9

10

8/1/04 10/31/04 1/30/05 5/1/05 7/31/05 10/30/05


Electrical Conductivity ( μS/cm)

200

300

400

500

600

700

800

900

8/1/04 10/31/04 1/30/05 5/1/05 7/31/05 10/30/05

HRC injection

Pumping


-20

-10

0

10

20

30

40

50

60

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

d1

3C

DIC

45 ft49.5 ft52.5 ft56.5 ft

d13C of HRC

d13C of Background

d13C of Dissolved Inorganic Carbon is Byproduct of HRC Metabolism

Biogeochemical Evidence of Microbial Metabolism in Groundwater


0

20

40

60

80

100

120

140

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

Acetate (ppm)

45 ft49.5 ft52.5 ft56.5 ft

0

20

40

60

80

100

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

Nitrate (ppm)

0

20

40

60

80

100

120

140

160

180

200

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

Sulfate (ppm)

Sulfate reductionSulfate reduction

Nitrate decrease Nitrate decrease

Biogeochemical Evidence of Microbial Metabolism in Groundwater

Acetate IncreaseAcetate Increase

1.E-04

1.E-03

1.E-02

1.E-01

1.E+00

1.E+01

1.E+02

8/2/04 9/1/04 10/2/04 11/1/04 12/2/04 1/1/05 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) Concentration, mg/L

44 pump4549.552.556.5Well 43

Fe(II) IncreaseFe(II) Increase


Changes of Cr(VI) Concentration in Groundwater after HRC Changes of Cr(VI) Concentration in Groundwater after HRC InjectionInjection

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

CrVI concentration (ppm)Injection well



Average Total Chromium 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

Total chromium (ppm)

Injection well




Approach

• Statistical analysis & data reduction used to mine vast quantities of data

• Organisms assigned to groups based on covariance (similar response to treatment)

• Combined with geochemical data and PLFA this yields insight into functional role/niche

of specific organisms

Combined High Density Microarray Analysis and Combined High Density Microarray Analysis and 1313C Phospholipid AnalysisC Phospholipid Analysis

Days since HRC injection

0 11 17 27

Corrected hybridization intensity

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


Low High

Hierarchical clustering and heatmap plot of 16S GeneChip analysis of microbial community sub-families detected during chromate bioremediation. PCA groups are indicated by brackets.

1

2

3

4

Bacteria and Archaea Detected

Grouped according to response to HRC during chromate remediation

Group1 organisms declinePseudomonas, Burkholderia (Denitrifiers) Acidithiobacillus, Thiothrix (Sulfur oxidizers)Leptothrix (Iron oxidizer)

Group2 organisms increase then declineAcidovorax, Thauera (denitrifiers)Flavobacteria (aerobes, use glycerol)

Group3 organisms decline then returnMainly oligotrophic bacteria

Group 4 organisms increase in late stagesLegionella, Chlamydophila, Flectobacillus.

High Density Microarray Analysis


Euryarchaeota(Methanogens)

Intensity (a.u.)

0

1000

2000

3000

45ft 49.5ft 52.5ft 56.5ft

0

1000

2000

3000

Injection well Monitoring well

9 17 216 312 9 17 216 312

Days post HRC Injection Days post HRC Injection

Comment – methanogenic conditions 312 days after single HRC injection


Intensity (a.u.)

0

1000

2000

3000

45ft 49.5ft 52.5ft 56.5ft

Intensity (a.u.)

0

1000

2000

3000

45ft 49.5ft 52.5ft 56.5ft

Bacteroidetes(Flavobacteriaceae)


9 17 216 312 9 17 216 312


Comment - Initial enrichment of Flavobacteria but declining over timeDo not use lactate – but use glycerol – hence no 13C detected in their PLFAs


Intensity (a.u.)

0

1000

2000

3000

45ft 49.5ft 52.5ft 56.5ft

Intensity (a.u.)

0

1000

2000

3000

45ft 49.5ft 52.5ft 56.5ft

Deltaproteobacteria(Desulfovibrionaceae)


9 17 216 312 9 17 216 312


Comment – continuous presence of Desulfovibrio – may help maintain chromium reducing conditions by producing H2S - observed


Intensity (a.u.)

0

1000

2000

3000

45ft 49.5ft 52.5ft 56.5ft

Intensity (a.u.)

0

1000

2000

3000

45ft 49.5ft 52.5ft 56.5ft

Deltaproteobacteria(Geobacteraceae)


9 17 216 312 9 17 216 312


Comment – continuous presence of Geobacter – may help maintain chromium reducing conditions by producing Fe(II) – observed also


13C Phospholipid Analysis

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

• 13C polylactate used as spike it is not esterified to glycerol backbone• it 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)


Major Findings to Date Despite low initial microbial densities (<105 cells g-1), HRC injection in

the groundwater stimulated increase in the biomass up to 107 - 108 cells ml-1

Highly reducing conditions were achieved quickly with hierarchical depletion of electron acceptors O, NO3, and Fe (III) (SO4 was reduced but never depleted except transiently months later), sulfate reduction has been sustained to for the last 20 months

SIP analysis confirmed microbial metabolism of HRC and PLFA indicated which group of organisms was utilizing the electron donor

Geophysical measurements were capable of characterizing hydrogeological conditions and monitoring the HRC distribution in groundwater

Biostimulation has not yet had an effect on subsurface flow

Cr(VI) was reduced to drinking water standards after increases in Fe(II), and has remained low for the last 20 months.

Microbial community structure changes indicate dominance by sulfate reducers and iron reducers that are apparently maintaining Fe(II) and Cr(VI) reduction


Future ResearchFuture Research

Well 699-96-45Well 699-96-44

• Metagenome Sequence by JGI• Metagenome (large Insert and

small insert clone libraries using MDA) by Diversa

• Isolation and sequencing of Desulfovibrio strains by JGI in the Lab Sequencing Program

• Mass transfer between high and low permeability zones

• Changes in hydraulic properties of sediments after HRC injection

• Evaluation of the potential for Cr(III) reoxidation

• Development of a numerical code TOUGH Bio-React

• Monitoring and new field tests (2 new wells in May).

Coring


Contacts and PublicationsDr. Terry C. Hazen [email protected]

Hanford Project http://esd.lbl.gov/ERT/hanford100h/

ERSP http://www.lbl.gov/ERSP

Hazen Lab http://www-esd.lbl.gov/ECO/Hazenlab/index.htm

Ecology Department http://www-esd.lbl.gov/ECO

Center for Environmental Biotechnology http://www-esd.lbl.gov/CEB

Virtual Institute for Microbial Stress and Survival http://vimss.lbl.gov

Dr. Terry C. Hazen [email protected]

Hanford Project http://esd.lbl.gov/ERT/hanford100h/

ERSP http://www.lbl.gov/ERSP

Hazen Lab http://www-esd.lbl.gov/ECO/Hazenlab/index.htm

Ecology Department http://www-esd.lbl.gov/ECO

Center for Environmental Biotechnology http://www-esd.lbl.gov/CEB

Virtual Institute for Microbial Stress and Survival http://vimss.lbl.gov

Earth Sciences Division, Ecology Department Field Investigations of Lactate-Stimulated Bioreduction...

Documents

Transcript of Earth Sciences Division, Ecology Department Field Investigations of Lactate-Stimulated Bioreduction...