Synchrotron-based investigations of biogeochemical transformations of priority contaminants in soils at DOE sties July 18, 2006 Presented by Jeff Fitts

Synchrotron-based investigations of Synchrotron-based investigations of biogeochemical transformations of priority biogeochemical transformations of priority

contaminants in soils at DOE stiescontaminants in soils at DOE sties

July 18, 2006

Presented by Jeff Fitts

Environmental Research & Technology Division

http://web.em.doe.gov/em94/sitesum.html

• 3,000 inactive waste sites;

• DOE EM (Environmental Management) FY06 appropriation $6.6 billion

• The Big Three: Hanford, WA; Oak Ridge, TN; Savannah River, SC

DOE Environmental Management Sites

J Fitts

Most of the smaller sites were previously used by the Department of Energy's predecessor agencies for research and production, or to mine and mill uranium. They are managed by the Formerly Utilized Sites Remedial Action Program (FUSRAP) and the Uranium Mill Tailings Remedial Action Project (UMTRA), respectively. The FUSRAP program is based at the Department's Oak Ridge office in Tennessee, and program activities across the country are funded through that office. The UMTRA program is funded through the Albuquerque office in New Mexico.

Research funding from ERSP within DOEEnvironmental Remediation Sciences Program mission is to advance our understanding of the fundamental biological, chemical, and physical processes that control contaminant behavior in the environment in ways that help solve DOE’s intractable problems in environmental remediation and stewardship.

ERSP Field Research Center at ORNLS-3 disposal ponds• U processing waste• In-use 1951-1983

http://www.lbl.gov/NABIR/

• Neutralized 1984• Paved 1988

J Fitts

The goal of the Natural and Accelerated Bioremediation Research (NABIR) Program is to provide the fundamental science to serve as the basis for the development of cost-effective bioremediation of radionuclides and metals in the subsurface at DOE sites. The focus of the program is on strategies leading to long-term immobilization of these contaminants in place, to reduce the risk to humans and the environment. The NABIR program encompasses both intrinsic bioremediation and accelerated bioremediation through the use of biostimulation (the addition of inorganic or organic nutrients). The program consists of four closely interrelated Science Elements, three Cross-Cutting Elements, and the Field Research Center (FRC). The Science Elements are Biogeochemistry, Biotransformation, Community Dynamics/Microbial Ecology, Biomolecular Science and Engineering. Cross cutting elements include Bioremediation and its Societal Implications and Concerns (BASIC), Integrative Studies, and Assessment (innovative method development). The NABIR program has established a Field Research Center on the U.S. Department of Energy Oak Ridge Reservation in Oak Ridge, Tennessee. The FRC is a focal point for integration of process level understanding and field research for the NABIR program. Scientific disciplines represented in the program include microbial physiology and ecology, molecular biology, geochemistry, hydrology, and mathematical modeling. Interdisciplinary research is strongly encouraged. The NABIR program does not support research on organic contaminants (with the exception of metal-chelator complexes) or on phytoremediation.

Manipulate Uranium oxidation state to control U solubility

• Oxidized uranium, U(IV), is soluble and mobile

• Reduced Uranium, U(IV), is insoluble and immobile

• Soil Bacteria often control the redox state of soil

Two remediation approaches

• Extract uranium from the ground

• Stabilize uranium in the ground

Groundwater Flow

Uranium Plume

1. High nitrate (~1000 ppm in Area 2)

2. Acidity

3. Heavy metals (Ni, Al…)

FRC conditions present challenges to in-situ FRC conditions present challenges to in-situ bioremediation strategies bioremediation strategies

Will introduction of nickel resistance into indigenous microbial community have a positive effect on nitrate reducers and stimulate iron and sulfate reducers?

Problematic conditions

1. Too high redox for stimulating sulfate and Fe reducers

2. Affects metal bioavailability, and thus, toxicity

3. Inhibit nitrate reducers

Result

J Fitts

WATERSHED COMMUNITY CHARACTERIZATION UPDATE - SHIFTS INGROUNDWATER MICROBIAL COMMUNITY STRUCTURE OBSERVED ALONGCONTAMINANT GRADIENTS (YAN, GENTRY, WATSON, AND ZHOU)Microbial communities were compared by Yan, Gentry, and Zhou in 6samples along contaminant transport pathways in shale (FW106,FW413, GW537, and PTMW02) and gravel substrates (FW231 andFW410) in addition to an uncontaminated background sample (FW301).Genomics DNAs were isolated from groundwater, purified, amplifiedwith primers 27F and 1492R, and cloned to produce 16S rDNAlibraries. Over 1,500 clones were sequenced, and data was analyzedusing DOTUR. The most highly contaminated groundwater, FW106 andFW413 (pH ~3.7; ~2,000 mg nitrate/l; ~20 mg uranium/l), had limiteddiversity with Shannon Index values of 0.66 to 1.33 compared to 4.89at the background site. Diversity values increased along thecontaminant gradient in the shale pathway with decreasedcontaminant levels. Diversity values were higher in the gravel pathway(~3) which also had higher pH and lower contaminant levels. Clonesfrom the highly contaminated groundwater in the shale pathway weredominated by γ-Proteobacteria, primarily an unclassifiedXanthomonadaceae related to Frateuria spp. The proportion of thisorganism was directly correlated with the level of contamination. Lesscontaminated groundwater along the shale and the gravel pathwaycontained communities strikingly different than the highlycontaminated groundwater. Our results suggest that microbialcommunity composition at the FRC is strongly influenced bycontaminant (i.e., nitrate, uranium) and pH levels. Further dataanalysis is being performed to determine the primary determinant(s)of community composition at the FRC. Additional samples from eachpathway along with samples from limestone, surface water, and springsare also being analyzed.In addition, Kerkhof, Kostka, and McGuinness are assaying microbialpopulations using DNA-based (presence/absence) measurements byTRFLP of 16S rRNA genes. These results will be summarized in a future update but results so far are consistent with the above findings.

ERSP project overviewERSP project overview

S3 ponds at ORNL

Goal: Immobilize uranium in contaminated sediments via microbial reduction and precipitation

Problem: Active uranium reducers are inhibited by co-contaminants in complex waste streams (e.g., heavy metals)

Major project objectives

Demonstrate application of natural gene transfer to improve community function under increased levels of toxic metal stress (Dr. Daniel van der Lelie, BNL Biology Dept.)

Demonstrate ability to enhance uranium immobilization in ORNL sediments by indigenous microorganisms that have adopted the toxic metal resistance marker (Fitts)

Project design schematicProject design schematic

FRC soils

Model organisms

Isolated from FRC fluidized bed reactor

Nickel stress1. Community structure2. Improved nitrate red.

Total community

Horizontal gene transfer (in vivo) – soil columns

Ni resistance

Strain construction(in vitro)

1. S and Fe reduction2. Uranium reduction and precipitation

mechanisms

What is really happening to the uranium?What is really happening to the uranium?

• How stable is the uranium associated with the soil?• Could it become a problem in the future?

Contaminant speciation in complex real-world systemsContaminant speciation in complex real-world systems

• Heterogeneous materials such as soils• Low contaminant concentrations

Single-crystal oxide/water interfacesSingle-crystal oxide/water interfaces

• Derive atomic-level picture• Test predictive models

Speciation on model soil particlesSpeciation on model soil particles

• Focus on predominant processes• Molecular details of transient species

Improve Improve predictions of predictions of contaminant contaminant behaviorbehavior

and in pure bacterial culturesand in pure bacterial cultures

1

2

3

1. Growth media

2. Common soil bacteria, Pseudomonas

w/ and w/o gene for Ni resistance

3. As a function of Ni concentration

Role of soil bacteria and their mechanisms of toxic Role of soil bacteria and their mechanisms of toxic metal resistancemetal resistance

Pure culture

1. Monitor bacterial growth (UV-Vis)

2. Measure Ni uptake by bacteria

2-5 l sample

X-ray transparent window

C=O

Scanning Transmission X-ray Microscope imaging at the carbon K-edge

Washed cells exposed to Ni for 2 hrs

Rinsed cells are dried on microscope window

Ni resistance mechanismsNi resistance mechanisms

Carbonates not observed

O K-edge will be sensitive to NiO formation

0.8m

2.5m

Carbonate in organic matrix

Pseudomonas DMY2::ncc-nre exposed to 2 mM NiCl2

C=C

carbonate

Optical Density at 290eV

Cluster image

NSLS Beamline X1A

U(VI) reduction by Fe(II)-containing minerals

5 nm

C

200 nm

Green rust:Fe(II)-containing mineral

Green rust afterRxn w/ U(VI)-containing aqueous solution

J Fitts

GRs typically form under near neutral to alkaline conditions in suboxic environments and have been identified as products of both abiotic and microbially induced corrosion of iron and steel as well as microbially mediated and abiotic reductive dissolution of ferric oxyhydroxides by Fe2+. Direct evidence for the presence of GRs in hydromorphic soils has recently been reported (Trolard et al. 1997; Abdelmoula et al. 1998; Génin et al. 1998) and there is evidence to suggest that GRs may control Fe solubility in these soils (Bourrié et al. 1999). In addition, GRs are metastable intermediates in the transformation of Fe(II) to magnetite and Fe(III) oxyhydroxides (e.g. lepidocrocite and goethite) under near neutral to alkaline conditions and are believed to play a central role in the redox cycling of Fe in many aquatic and terrestrial environments.

Remobilization potential of sequestered UraniumRemobilization potential of sequestered Uranium

After initial rxn with Green rustAfter initial rxn with Green rustReduced U, sequestered as U(IV)Reduced U, sequestered as U(IV)

After exposure to oxygenAfter exposure to oxygenMixture of U(VI) & U(IV)Mixture of U(VI) & U(IV)

Aqueous U(VI)Aqueous U(VI)Bioavailable UraniumBioavailable Uranium

How stable is U(IV) that is associated with Fe-oxide?How stable is U(IV) that is associated with Fe-oxide?

What is the role of aqueous Fe(II)?What is the role of aqueous Fe(II)?

Pseudomonas DMY2 tested in column studiesPseudomonas DMY2 tested in column studies

Media +Formaldehyde

Media +1 mM NiCl2

Mediano Nickel added

1 2 3 4 5 6 7 8 9Kill

2,6 - FRC community3,7 - FRC community + Pseudomonas wild type4,8 - FRC community + Pseudomonas pMol2225,9 - FRC community + Pseudomonas::ncc-nre

Geochemical interrogation: S, Fe & U at time zeroGeochemical interrogation: S, Fe & U at time zero

U oxidation state at M5 edge

S speciation and redox state

U-Fe correlationU distribution

port B

Typical soilOrganic matter

Area 2 FRC soil

inorganic sulfatesulfate

reduced organic S species

Area 2 FRC soil

U6+ standard

U4+ standard

NSLS beamlines X27A & X15B

Geochemistry of columns after 65 daysGeochemistry of columns after 65 days

Ni distribution in Column 2

•No reduction of Uranium observed

•Small increase in sulfide relative to kill (oxidation during transfer may be problem)

•Fe(III) oxides still dominate

•Initial mobilization of Uranium

•Nickel breakthrough observed but significant adsorption occurs

Soil indicators by x-ray absorption spectroscopy

Column effluent indicators

• Determine chemical state of untreated Determine chemical state of untreated wasteswastes

• Assess and optimize treatment technologies Assess and optimize treatment technologies and remediation strategiesand remediation strategies

• Assess end-states to improve long-term Assess end-states to improve long-term performance predictionsperformance predictions

• Conduct basic science research to seed Conduct basic science research to seed future innovation in cleanup technologies future innovation in cleanup technologies

Summary: Applications of synchrotron-based Summary: Applications of synchrotron-based studies in nuclear waste managementstudies in nuclear waste management

Acknowledgements

NSLS Measurements Paul Northrup (BNL Environmental Sci. Dep.) – XAS & MicroprobeBjorg Larson, Sue Wirick (Stony Brook University) – STXMJames Ablett (BNL NSLS) – Microprobe beamline X27A

Oak Ridge FRCDave Watson (ORNL)

BNL Biology DepartmentNiels van der LelieDavid Moreels, Safiyh Taghavi, Craig Garafola

BNL Environmental Sciences Dept.Garry Crosson

Documents

Synchrotron-based investigations of biogeochemical transformations of priority contaminants in soils at DOE sties July 18, 2006 Presented by Jeff Fitts