LA-UR-

Researchers at LANL are demonstrating a multi-layered permeable reactive barrier (PRB), for in situ treatment of groundwater bearing mixed contaminants. A biobarrier layer removes dissolved oxygen and promotes anaerobic microbial reduction of nitrate and perchlorate. The location in Mortandad Canyon was selected because of the directed flow of alluvial groundwater, a large network of existing monitoring wells, and a well-documented contaminant history. The PRB uses a funnel-and-gate system with four sections to immobilize or destroy contaminants, including perchlorate, nitrate, 90Sr, 238,239,240Pu, and 241Am. The four cells, ordered by sequence of contact with the groundwater, consist of a colloid barrier, a mineral apatite layer, a biobarrier, and a “polishing” layer. The biobarrier maintains a one-day average residence time. Four monthly data sets were collected. Collaborators at the University of California at Berkeley conducted a series of microbial analyses to evaluate function of the biobarrier. Water was obtained from 3 sampling wells across each cell, and from wells located up- and down-gradient. Overall, results show microbial reduction of nitrate and perchlorate to levels below detection. Field parameters include DO, pH, redox (ORP), and others. The laboratory microbial methods include enumeration by Most Probable Number (MPN) analysis of nitrate- and perchlorate-reducers, assays for metabolite organic acids (acetate, propionate, etc.), ferrous/ferric iron, DNA-specific probes for known perchlorate-reducer genera, and a new RNA-based assay for chlorite dismutase activity. Additional results show moderate numbers of both dissimilatory perchlorate- and nitrate-reducing populations, production of acetate and propionate, and reduced DO and pH levels. The dominant group of perchlorate reducers present was from the previously described Dechloromonas genus, in the beta subclass of the Proteobacteria, which together with Dechlorosoma are considered to be the primary genera in circum-neutral mesophilic environments. The performance of the PRB will be evaluated through its lifetime, projected to be ~10 years. Final report of PRB installation can be found at URL: http://www-emtd.lanl.gov/P2/Barrier/Mortendad.html.

ABSTRACT:

Principles of Microbial Perchlorate Reduction

• Perchlorate-reducing bacteria are apparently ubiquitous in soil• Process enzymatically reduces perchlorate to chlorate, then

chlorite, with subsequent dismutation to chloride and oxygen• Organisms “respire” using perchlorate as an electron acceptor in

place of oxygen or nitrate, organic carbon acts as electron

donor• Microorganisms are facultative anaerobes from several different

genera, primarily Dechloromonas and Dechloromusa• Nitrate can be an inhibitor of perchlorate reduction, mechanism

of inhibition is unclear, although is probably related to redox

potential• Anoxic conditions required for perchlorate reduction, redox

potential required for reduction to proceed is lower than for

nitrate reduction

Principles of Biological Denitrification

• “True” denitrification is a reductive process that yields energy for microbial cell growth

• Process sequentially reduces nitrate to nitrite, nitric oxide, nitrous

oxide and nitrogen gas• Organisms “respire” using nitrate as an electron acceptor in

place of oxygen, organic carbon acts as electron donor• Microorganisms are facultative anaerobes from many different

genera, e.g. Pseudomonas, Bacillus, etc.• Both carbon and phosphorus may be limiting in natural ground

waters, preventing the timely destruction of nitrate

Introduction

• Perchlorate and nitrate are growing worldwide problems, existing as highly mobile anionic groundwater contaminants = large plumes• Biological reduction of perchlorate and nitrate in groundwater is

under development as a technology to address these problems• EPA drinking water limit may be as low as 1 цg/L (ppb) perchlorate• Maximum Contaminant Level (MCL) is 10 mg/L nitrate-N• Perchlorate contamination is primarily from DoD propellant production and disposal (ammonium perchlorate)• Major sources of nitrate are over-fertilization, animal farms/dairies

and nitric acid use in mining and weapons programs• Use of nitric and perchloric acid at LANL without removal during

water treatment resulted in significant groundwater contamination

(Per)chlorate Reduction Reactions(Per)chlorate Reduction Reactions Microbial Denitrification ReactionsMicrobial Denitrification Reactions

• Overall metabolic reduction reaction:

3Ac- + 4ClO3- + 3H+ -----> 6CO3

- + 4Cl- + 6H2O

*Ac = acetate

• Enzymatic reduction reactions:

ClO4 ClO3

ClO2 Cl + O2

(per)chlorate reductase* chlorite dismutase

([per]chlorate) (chlorate) (chlorite) (chloride)

*Both (per)chlorate and chlorate appear to be substrates of the same enzyme

• Metabolic denitrification reaction:

0.625Ac- + NO3 -----> 1.25HCO3 + 0.25 N2

• Cell component synthesis reaction:

3.5Ac + NO3 -----> C5H7O2N + 2HCO3

• Combined reaction:

0.712Ac + NO3 -----> 0.485N2 + 0.03C5H7O2N + 1.273HCO3

Ac = acetate; C5H7O2N = representative cellular material composition

AtmosphericNitrogen

Organic nitrogen

Ammonium

NitrogenFixation

Nitrite

Nitrate

Ammonification

Oxidation

Fertilizer

Nitrate Contamination

Water TableLeaching

Plant Uptake

Plant Decay

Animal &HumanWaste

Industry

AtmosphericNitrogen

Ground WaterGround Water Nitrate Contamination

Oxidation

Denitrification

N2 Gas

The Environmental Nitrogen Cycle

Multiple LayeredMultiple Layered In Situ In Situ PRB for PRB for Groundwater RemediationGroundwater Remediation

0

0.5

1

1.5

2

2.5

0 10 20 30 40 50 60 70 80 90 100 110

Time (days)

Sr

an

d C

lO4

Con

c. (

mg/

L)

0

20

40

60

80

100

120

140

NO

3 C

onc.

(m

g/L

)

Sr data

ClO4 (mg/L)

NO3 data

PRB Laboratory ResultsPRB Laboratory Results

• Designed to remove radionuclides (e.g. Sr, Am, Pu, U), metals (e.g. Pb, Co, Cr), nitrate and perchlorate

• Four sections with unique purpose: colloid barrier, apatite, biobarrier, gravel polishing section

• Bench-scale batch studies, column studies in 1- and 2-dimensions to demonstrate effectiveness of each section

• Bench-scale mock-up of full-scale system in final design sequence, actual groundwater used over ~one year period

• Demonstration PRB installed in January 2003

PRB Laboratory Mock-upPRB Laboratory Mock-up

Microbiology of a Biobarrier for In Situ Remediation of Nitrate and PerchlorateB.A. Strietelmeier1*, J.D. Coates2, J. Pollock2, J.P. Kaszuba1, T.P. Taylor1, and P. Longmire1

1Los Alamos National Laboratory Session 260 *Corresponding author: Phone: (505) 665-99862University of California, Berkeley Poster Q-338 Fax: (505) 665-4955, E-mail: bastriet@lanl.gov

LA-UR-04-3226

Microbial Characterization of PRB Water SamplesMicrobial Characterization of PRB Water Samples

Monitoring ResultsMonitoring Results

PRB Design – Funnel and GatePRB Design – Funnel and Gate

Wing WallsSamplingPoints andWellsGroundwaterFlowScoria Gravel (colloids)

Apatite

(disso

lved

actinit

es, me

tals)

~ 24 feet17feetRemediatedGroundwaterBio-Barrier (nitrate,

perchlo

rate)

Limeston

e Grave

l(pH control)

GroundwaterFlowGApBioLs~ 24 feet27feetgradeBedrock10feet3feet

PRB Installation –Post Construction, Wing WallsPRB Installation –Post Construction, Wing Walls

View of Completed PRB

Wing Wall Installation

Detailed Hydrologic Cross-Section of PRBDetailed Hydrologic Cross-Section of PRB

Hydrogeologic cross-section from west to east through the PRB and PRB monitor wells 1 and 2 in Mortandad Canyon. The contact between bedrock and alluvium and the bottom of the PRB are also shown. Only the lowermost portion of the PRB is depicted, as elements extend to ground surface at an elevation of 6882 feet. The positions of individual cells within the PRB are shown schematically. The elevation of groundwater in the alluvial aquifer for mid-May through mid-September is plotted. Groundwater levels lying below the alluvium-bedrock contact are interpreted as residual groundwater in well bottoms.

6845

6847

6849

6851

6853

6855

6857

150 160 170 180 190 200

Distance from Well MCO-4b (feet)

Elevation (msl)

May

June

July

August

SeptemberBedrock/Alluvium Contact

PRB Profile

PRB-MW-01

PRB-MW-02

Gravel (Scoria)

Cell

Apatite CellBiogeochemical

Cell

(Biobarrier)Limestone Cell

residual water in dry well,

September

residual water in dry well, August

and September

AcknowledgementsAcknowledgements

• Ferrous iron production in the biobarrier indicates iron-reducing conditions are present• Dominant group or perchlorate reducers was Dechloromonas, and members were most closely related to D. aromatica, strain RCB• Continued monitoring, potentially including tracer studies, is

necessary to understand the system more fully• A scientific study of the Mortandad Canyon PRB would be of great benefit to assist in the understanding of similar systems at other sites, funding is being pursued for such a study• A lot more needs to be learned about the radionuclide and metal interactions with the PRB materials

• Metabolic by-products: organic acids - acetate, propionate, butyrate

• IC analysis of full suite of anionic species

• Total ferric and ferrous iron• Identification of dominant perchlorate-reducing species• Test for nitrate reduction by perchlorate-reducing species

Microbial Analyses Microbial Analyses ConductedConducted

• Initial results from monitoring and microbial studies in May and August 2003 show that reduction of perchlorate and nitrate is occurring in the apatite and biobarrier sections of the PRB• Results also indicate that insufficient water was present in this time period for appreciable flow to occur through the PRB• MPN results indicate that both ClRB and DNB populations are increasing in the biobarrier during this time period• Measures that indicate viable populations include reduction

of oxygen, perchlorate and nitrate, production of metabolic by-products, and decrease in pH and ORP• A multi-layered PRB designed to remove Pu, Am, Sr, nitrate

and perchlorate from flowing alluvial groundwater has been successfully emplaced in Mortandad Canyon at LANL

• Department of Energy (DOE) Pollution Prevention program for funding PRB development and installation• DOE Fire Recovery program for partial funding of installation• Shaw Environmental and Infrastructure, Inc. for design and installation, hydrologic modeling and field support• Laboratory studies team – S.D. Ware, P.A. Leonard,

E.M. Hodge, B.A. Martinez, M.L. Espinosa and J.D. Adams, N. Lu, J. Conca and J. Wright• University of California, Berkeley, Department of Plant and Microbial Biology• Ernest Orlando Lawrence Berkeley National Laboratory, Earth Sciences Division

ConclusionsConclusions

UC Berkeley team performed full characterization of two sample sets, 5/03 and 8/03

Identification of Dominant Perchlorate-Reducing Species

Results – May 2003 and August 2003*

Site Name

Universal Primers (control)

WD Primers

CKB Primers

RCB Primers

PS Primers

PRB (1)-2 (+) (−) (−) (+) (−)

(1)PRB -3 (+) (−) (−) (+) (−)

(1)PRB -4 (+) (−) (−) (+) (−)

(1)PRB -7 (+) (−) (−) (+) (−)

(+) 16 S DNA was successfully a .mplified (-) 16 S DNA wasnot .amplified

*Results were the same for both May and August 2003

0

0.2

0.4

0.6

0.8

1

1.2

Fe Concentration, mmol/L

Downgradient Upgradient Bio Center Ap center G center Bio north

Well Location

Ferrous and Ferric Iron in Water Samples from May and August 2003 in Mortandad Canyon PRB

Fe2+ May

Fe2+ Aug

Fe3+ May

Fe3+ Aug

0

0.5

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1.5

2

2.5

3

3.5

4

Concentration, mmol/L

Downgradient Upgradient Bio Center Ap center G center Bio north

Well Location

Production of Metabolic Products in Water Samples from May and August 2003 in the Mortandad Canyon PRB

Acetate May

Acetate Aug

Propionate May

Propionate Aug

0.1

1

10

100

1000

10000

Cells/ml

Downgradient Upgradient Bio Center Ap center G center Bio north

Well Location

Most Probable Numbers of Nitrate- and Perchlorate-Reducing Bacteria in Water Sampled in May and August 2003 from Mortandad Canyon PRB

N - May (x10^6)

N - Aug (x 10^6)

C - May (x 10^1)

C - Aug (x 10^1)

,

pH and Dissolved OxygenpH and Dissolved Oxygen Perchlorate (ClOPerchlorate (ClO44--), Chlorate (ClO), Chlorate (ClO33

--), Chlorite ), Chlorite

(ClO(ClO22--), and Bromide (Br ), and Bromide (Br --) Concentrations) Concentrations

Monitoring Wells in PRBMonitoring Wells in PRB

1

10

100

1000

10000

MCO-4B GRAVEL APATITE BIO BARRIER LIMESTONE MCO-5

WELL OR PRB CELL

Figure C-2.Distributions of perchlorate (ClO4-1), chlorate (ClO3-1), chlorite (ClO2-1), and bromide (Br) in wells and in the PRB installed in Mortandad Canyon. Detection limits (DL) for ClO4-1, ClO3-1, and ClO2-1 are 2, 10, and 100 ppb, respectively, using ion chromatography.

Br (08/03)

ClO2 (08/03)

ClO3 (08/03)

ClO4 (08/03)

Br (07/03)

ClO2 (07/03)

ClO2 (05/03)

ClO3 (07/03)

ClO3 (05/03)

ClO4 (07/03)

ClO4 (05/03)

Mortandad Canyon LocationMortandad Canyon LocationMortandad Canyon Mortandad Canyon In SituIn Situ Perchlorate And Nitrate Perchlorate And Nitrate Reduction DemonstrationReduction Demonstration

2D Mock-up of PRB Design

Site Characteristics and Design ParametersSite Characteristics and Design Parameters

Design Parameters• Target alluvial groundwater, minimize surface erosion/infiltration • Optimize hydraulic capture, minimize reactive volume• Minimize excavated soil requiring waste disposal• Residence time in biobarrier, 1 day minimum• Lifetime = ~10 years• Install ports for access to solids and aqueous fluids

NSBedrock(Volcanic Tu ff)AlluviumSurfaceWaterFlowHydraulicFloor Ground WaterFlow Not to Scale

Mortandad Canyon• Hydrologic funnel• Hydraulic floor• Groundwater flow

surface water flow• Low-level rad in soil• Geotechnically

feasible

• Demonstration multi-layered PRB system installed in Mortandad Canyon in path of large plume resulting from contaminated water discharged from wastewater treatment plant

• Wastewater plant treatment system not designed initially to remove perchlorate and nitrate, discharged for over 40 years

• Recent addition of ion exchange system has removed the perchlorate source contamination

• PRB operational since January 2003, drought has resulted in low water conditions and no flow through PRB for most of this period

• Monitoring began in May 2003 and continues to present