Pedro J.J. Alvarez, Ph.D., P.E., DEE

Iron-Based Bioremediationof Aquifers Contaminated with RDX or Other Oxidized PollutantsINCA, Venice, September 2, 2004

Acknowledgments

Students• Dr. Joshua Shrout

• Dr. Byung-Taek Oh

• Dr. Craig Just

• Dr. Jose Fernandez

• Dr. Kevin Gregory

• Mr. Brett Sutton

• Mr. Phil Larese

• Ms. Leslie Sherburne

• Mr. Sumeet Gandhi

Faculty• Michelle Scherer• Jerry Schnoor• Gene F. Parkin• Richard Valentine

Funding• SERDP

Wash water from munitions facilities was discarded in drains and lagoons.

Recalcitrant & toxic (seizures, rat poison, Class C carcinogen).

Its persistence and high mobility in aquifers represent a major remediation challenge at numerous military facilities.

RDX – A military explosive that has become an onerous groundwater pollutant

N

N

NNO2

NO2

O2N

RDXhexahydro-1,3,5-trinitro-1,3,5-triazine

UNEP POPs List (Dirty Dozen)

• Dioxins • Furans • PCBs • HCB • DDT • Chlordane • Toxaphene • Dieldrin • Aldrin • Endrin • Heptachlor • Mirex

Permeable Reactive Fe(0) Barrier

0

10

20

30

40

50

60

70

8019

70

1975

1980

1985

1990

1995

2000

Year

Pu

blic

atio

ns

Rel

ated

to

PR

B's

Recent Explosion of Activity Directed at Fe0 PRBs

OGI Database: http://cgr.ese.ogi.edu/ironrefs/

Time, daysTime, days

00 11 22 33 44 55

Co

nce

ntr

atio

n, m

g/L

Co

nce

ntr

atio

n, m

g/L

00

22

66

88

1010

1212

Reactivity of RDX and Other Pollutants with FeReactivity of RDX and Other Pollutants with Fe00

Reactors contained Master Builder’s FeReactors contained Master Builder’s Fe00 filings (4%, w/v), at 20 filings (4%, w/v), at 20ooCC

RDXRDX

NONO22--

NONO33--

TCE TCE

Cr(VI)Cr(VI)

Decreasing ReactivityDecreasing Reactivity

44

SEM Immage of Fe(0) Sample from a Barrier Treating a SEM Immage of Fe(0) Sample from a Barrier Treating a Chlorinated Solvent Plume in Kansas City.Chlorinated Solvent Plume in Kansas City.

Alvarez P.J.J et al., (1999). Proc. Bioremediation Research Program Review, Bloomingdale, IL EPA/600/R-99/092, p. 14-16

Fe0 Can Support Cell Growth Via H2 Production

0.000

0.005

0.010

0.015

0.020

0

10

20

30

40

50

0 2 4 6 8 10 12Time, days

Fe(0) Cells + NO3-

H 2

H2

NO3-

OD600

Nitr

ate

(mg/

L as

N)

P. denitrificans O

D at 600 nm

H2

Fe0 + 2H2O Fe+2 + H2 + 2OH-

Till et al., Environ. Sci. Technol., 1998, 634-639

AbioticAbioticReductionReduction

Microbial Reduction:Microbial Reduction:increases rate, less toxic productsincreases rate, less toxic products

Hypothesis 1Hypothesis 1HH22 produced by (anaerobic) iron corrosion stimulates produced by (anaerobic) iron corrosion stimulates

anaerobic bioremediation (e.g., RDX mineralization)anaerobic bioremediation (e.g., RDX mineralization)

RDXRDX MNXMNX, , DNX, TNX, othersDNX, TNX, others

CellCell

HH22

FeFe2+2+FeFe00

2 e2 e--

RDXRDX

COCO22, CH, CH44, other?, other?

HH22

HH22

FeFe00 + 2H + 2H22O FeO Fe+2+2 + + HH22 + 2OH + 2OH--

RDX Mineralization (14CO2) is mediated by bacteria, and Fe0 has a stimulatory effect

Time (days)

0 15 30 45 60 75

Cu

mu

lati

ve 14

RD

X M

iner

aliz

atio

n (

%)

0

20

40

60

80

100

Sterile Fe0

Soil + sludgeSoil + Fe(0) + sludge

Oh, Just, and Alvarez (2001). Environ. Sci. Technol. 35(21):4341-4346

k, day-1

H2 supply limits

Effect of Fe0 quantity on SRB Activity

Fe0 + 2H2O Fe2+ + 2OH- + H2

Fe0 surface area concentration, m2/L

0 1000 2000 3000 4000 5000

Su

lfat

e re

mo

val r

ate

coef

fici

ent,

k (

day

-1)

0.0

0.1

0.2

0.3

0.4

0.5

6

7

8

9

pH

High pH limitation

0 1000 2000 3000 4000 50000.0

0.1

0.2

0.3

0.4

0.5

6

7

8

9

pH

6

7

8

9

10

Fernandez-Sanchez, Sawvel, and Alvarez (2003). Chemosphere. 54 (7): 923-829.

Identification of Unknown IntermediateQuattro LC LC/MS/MS with a photodiode array detectorUsed acetate buffer and a scan range of m/z 61 to 300

Time, minutes

0 1 2 3 4 5

Tot

al I

on C

urre

nt

m/z50 100 150 200 250 300

Re

lativ

e I

nte

nsi

ty,

%

195

217

97

80

62

[M+59]-

10

0

N NO2N NO2

H H

MDNA

(methylenedinatramine)

N2O

(M

)

0

10

20

30

40

50

60

Soil Soil+

Fe0

Soil+

cells

Soil+

Fe0&cells

Fe(0) Surface Area Concentration (m2/L)

0 200 400 600 1000 1200

14C

-Lab

ele

d S

oil-

Bo

un

d R

esid

ue

(%

)

0

7

14

21

28

35

BioaugmentedPoisoned

Some 14C became soil-bound residue (Less in bioaugmented Fe0 treatments)

N

N

NNO2

NO2

O2N N

N

N

NO2

NOO2N N

N

NNO

NO

O2N N

N

NNO

NO

ON

NH

NH

NO2O2NHOH2C

NCH2OH

NO2

NHHOH2C

O2NH

NH

NO2

HCHO

N2O

HCOOH

CH4 CO2 CH3OH

N2

NHOH2C R

R'O

NHCH2OH

NO

+

RDX MNX DNX TNX

methylenedinitraminebis(hydroxymethyl)nitramine

hydroxymethylnitraminenitramine hydroxymethylnitrosamine

+acetogens

methanogens

amide linkage &sequestration byhumic backbone

H2O2 HCHO

3 H2O

2 H2O

H2O

3

3 HCHO 3

H2O3

Proposed RDX Degradation Pathway

Hypothesis 2Hypothesis 2

Dissimilatory iron(III)-reducing bacteria (DIRB?) could dissolve oxides that passivate the iron

surface, and generate reactive solids with surface-associated Fe(II) (e.g., green rust)

Cell

MNX, DNX,TNXMEDINAOthers?

Fe(III) oxide layerLactate

e-

Fe3+

MNX, DNX, TNX, HCHO,Others

Fe0

CellCell

RDX

Acetate

e-

RDX

Fe0Fe0

Fe2+

FeFe00

Reactive Biogenic Solid Produced by DIRB (Geobacter metallireducens GS15)

Initial oxide is inert Biogenic oxide degrades RDX

Time (hr)

0 100 200 300 400 500 600 700

RD

X C

on

cen

tra

tion

(M

)

0

10

20

30

40

50

Time (hr)

0 100 200 300 400 500 600 700

RD

X C

on

cen

tra

tion

(M

)

0

10

20

30

40

50

Fe(III) Oxide

After ~3 weeks

Gregory, Williams, Parkin, Scherer (2003). ACS Abstracts. 226: 063-GEOC Part 1 SEP 2003

80

60

40

20

0

mole

200150100500Time (min)

0

N2O

NH4+

HCHO

RDX

Green Rusts – Product Search

Abiotic degradation of RDX is fast – but . . .

1.0

0.5

0.0

RD

X (

C /

C0 )

5000

Time (h)

Biogenic ~ 5 months

Biogenic ~ 3 weeks High Fe(II)

Low Fe(II)

Green Rust

Flow-Through Columns

Evaluate RDX removal in columns mimicking PRBs under different microbial conditions (focus on long-term removal efficiency and permeability)

Columns packed with different layers

- Soil-Fe0-Soil, poisoned with a biocide (Kathon, 1 ml/L)

- Soil-Fe0-Soil (allowed colonization by indigenous cells)

- Soil-Fe0-Soil, bioaugmented (Shewanella algae BrY)

- Soil-Fe0-Soil, bioaugmented (G. metallireducens GS15)

- Soil-Fe0-Soil, bioaugmented with anaerobic sludge

- Soil-glass beads-Soil (control)

HRT = 0.5-2 d, Q = 6 ml/hr

30 c

mFe0

soil

soil

Flow-Through Columns—Simulated Permeable Reactive Iron Barrier

14C-RDX Concentration Profiles after 65 days(C0 = 18 mg/L and 10 Ci/L)

0 5 10 15 20 25 300.0

0.2

0.4

0.6

0.8

1.0

1.2

Distance from column inlet (cm)

0 5 10 15 20 25 300.0

0.2

0.4

0.6

0.8

1.0

1.2

Distance from column inlet (cm)

0 5 10 15 20 25 300.0

0.2

0.4

0.6

0.8

1.0

1.2

Soil Iron Sand

Soil Iron Sand Soil Iron Sand

Colonized (indigenous) column

BRY-bioaugmented column GS15-bioaugmented column

RD

X C

on

c.

(C/C

0)

0 5 10 15 20 25 30

RD

X C

on

c.

(C/C

0)

0.0

0.2

0.4

0.6

0.8

1.0

1.2Soil Iron Sand

14C-activity

RDX

Sterile Control

14C Concentration Profile in Naturally-Colonized Column(C0 = 16 mg/L and 10 Ci/L, HRT = 1.7 days , Operation time = 370 days)

Distance from Column Inlet (cm)

0 5 10 15 20 25 30

14C

Co

nc

entr

ati

on

(C

/C0)

0.0

0.2

0.4

0.6

0.8

1.0

1.2RDXMNXDNXTNXunknown1unknown 2unknown 3unknown 4Total Soluble 14C

Fe0 layer

14C Concentration Profiles in Column Bioaugmented with Shewanella algae BrY

(C0 = 16 mg/L and 10 Ci/L, HRT = 1.7 days, Operation time = 370 days)

Distance from Column Inlet (cm)

0 5 10 15 20 25 30

14C

Co

nc

entr

ati

on

(C

/C0)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

RDXMNXDNXTNXunknown1unknown 2unknown 3unknown 4Total Soluble 14C

Fe0 layer

14C Concentration Profiles in Column Bioaugmented with Geobacter metallireducens GS-15

(C0 = 16 mg/L and 10 Ci/L, HRT = 1.7 days, Operation time = 370 days)

Distance from Column Inlet (cm)

0 5 10 15 20 25 30

14C

Co

nc

entr

ati

on

(C

/C0)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

RDXMNXDNXTNXunknown1unknown 2unknown 3unknown 4

Total Soluble 14C

Fe0 layer

Distance from Column Inlet (cm)

0 5 10 15 20 25 30

pH

6

7

8

9

10

11

Control ColumnS. algae BrY ColumnG. metallireducens GS-15 Column

Montmorillonite (9%) buffers against pH increase,enhanced NO3

- removal, prevented NO2- accumulation

TIME, Days

0 5 10 15 20 25 30

0

10

20

30

40

pH = 10pH = 9

[NO

3- ], [

NO

2- ] m

g/L-

N

NO3- , no clay

NO2-

NO3- + clay

NO2-

Dejournett T. and P.J.J. Alvarez (2000). Bioremediation Journal 4:149-154.

Bioaugmentation Studies with Anaerobic Sludge for RDX Removal

(Co = 18 mg/L, 10 µCi/L HRT = 1.7 days)

Distance from column inlet (cm)0 5 10 15 20 25 30

14C

-Act

ivit

y (C

/Co)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Soil Iron filings Sand

Distance from column inlet (cm)0 5 10 15 20 25 30

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Soil Iron filings Sand

T = 20 days T = 390 days

Control

Poisoned column

Colonized column

Bioaugmented (sludge)

Throughput (Bed volumes)

0.0 0.5 1.0 1.5 2.0

C/C

0

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Throughput (Bed volumes)

0.0 0.5 1.0 1.5 2.0

C/C

0

0.0

0.2

0.4

0.6

0.8

1.0

1.2T = 0 monthsn = 0.552D = 1.321

T = 10 monthsn = 0.538D = 1.335

T = 10 monthsn = 0.474D = 1.041

T = 0 monthsn = 0.488D = 1.013

Colonized Column Bioaugmented Column

Bromide Breakthrough Curves Show No Significant Decrease in Porosity

cm2/h cm2/h

Co-contaminants were also removed

Distance from column inlet (cm)0 5 10 15 20 25 30

Co

nce

ntr

atio

n (

mg

/L)

0

2

4

6

8

10

12

Soil Iron SandSoil Iron Sand

Bioaugmented columnColonized column

Distance from column inlet (cm)0 5 10 15 20 25 30

Co

nce

ntr

atio

n (

mg

/L)

0

2

4

6

8

10

12HMX RDXTNT2,6DNT2,4DNT

Dominant species

A - Acetobacterium sp.B - Acetobacterium sp.C - Acetobacterium sp.D - Arthrobacter subdivision

Homoacetogens (A,B,C):

4H2 + 2CO2 CH3COOH + 2H2O

Ste

rile

Col

oniz

ed

Bio

augm

ente

d

DGGE Analysis of Microorganisms in Fe0 Samples

RDX MNX, DNX, TNX, others

Homoacetogen

H2

Fe2+FeFe00

2 e2 e--

RDX

CO2, CH4, other?

H2

H2

Hypothesis 3Homoacetogens participate directly or indirectly in RDX degradation

• Directly by possibly cometabolizing RDX with H2 as primary substrate

• Indirectly by comensalistically supporting heterotrophic activity, feeding DIRB (that dissolve or activate iron oxides) or other bacteria

that degrade RDX, possibly using it as an N-source.

Acetate

CO2FeFe00

Fe(III) oxide layer

Fe(II)(Surface)

Fe(III)

CO2, other?

e-

MNX, DNX, TNX, others

HH22

DIRB

CellsCells

RDX

RDX Removal by H2-Fed Homoacetogenic CultureIsolated from Bioaugmented Column

Time (day)

0.0 0.5 1.0 1.5 2.0

RD

X C

on

c. (

mg

/L)

0.0

0.4

0.8

1.2

1.6

2.0

Time (day)

0.0 0.5 1.0 1.5 2.0

Ace

tic

acid

Co

nc.

(m

M)

0

1

2

3

4

5

Culture medium

DI H2O

RDX as nitrogen source for Acetobacterium paludosum?

Natural Attenuation and Mineralization of RDX byproducts Downgradient of a PRB (soil HRT = 9 h)

Fe0

(~1 cm

HRT = 1h)

Soil

(15 cm

HRT = 9h)

1 ml/h

0

0.2

0.4

0.6

0.8

1

Influent Post Fe0 Post Soil

Port

C/C

0

DIC RDX

Fraction I Fraction II

SUMMARY OF RDX EXPERIMENTS

Showed high and sustainable RDX removal efficiency in biologically-active iron columns, and no clogging problems

RDX was mineralized in biologically-active treatments with Fe0, but not in sterile Fe0 systems.

Identified some key biogeochemical interactions- Biostimulation by H2 gas production during Fe0 corrosion- Homoacetogens as primary producers?- Production of reactive Fe(II) species by iron reducers

Demonstrated that both structural and adsorbed Fe(II) can reduce RDX via an abiotic pathway

Bioaugmentation with Methanogenic Consortium Enhanced CHCl3 Removal in Steel-Wool Columns

Weathers et al., Environ. Sci. Technol., 1997, 880-885

0.0

0.5

1.0

1.5

Eff

luen

t C

HC

l 3 (

M)

0.0

0.1

0.2

0.3

0.4

0 10 20 30 40 50 60 70

Time (days)

CH

4 (m

M)

Influent CF HRT = 2.4 d

Steel wool + cells

Steel wool alone

Combined Microbial-Fe(0) System Also Removed Nitrate Faster and Better

Till et al., Environ. Sci. Technol., 1998, 634-639

H2 & P. denitrificans Steel Wool & P. denitrificans

Steel Wool

Unreacted NO3-

Reduced by Fe(0) to NH4+

Unrecovered

Assimilated by bacteria

Denitrified by cells to N2

Cell Decreased the Activation Energy

0.20

0.15

0.10

0.05

Nitr

ate

Rem

oval

Rat

e C

oeff

icie

nt (

hr-1)

6050403020100

Temperature (oC)

Fe0 alone

Ea = 36.3 KJ/mol

Fe0 + cells

Ea = 24.3 KJ/mol

Batch, 70 g/L Masterbuilder® Fe0 filings + Paracoccus denitrificans (100 mg/l)

Ginner et al., (2004) Environ. Eng. Sci., 21: 219-229

Basis of Fe0-Bacteria Synergism

Fe0 corrosion rapidly induces anaerobic conditions that favor the degradation of oxidized pollutants

Bacteria remove passivating H2 layer from Fe0 surface, increasing e- flow from Fe0 (cathodic depolarization)

H2 enhances anaerobic bioremediation, which could offer alternative pathways leading to better end products

Removal of inhibitory compounds by Fe0 (e.g., RDX) enhances bacterial participation in cleanup and polishing

If dealing with mixtures, bacteria can remove compounds that Fe0 cannot (CH2CL2, sulfate)

Reductive dissolution of oxides (depassivation of iron surface) and microbial production of reactive surface-bound Fe(II) species (green-rust-like minerals)

Indigenous cellscolonize ZVI-PRB

Integrated ZVI-bioremediation system to intercept and degrade redox-sensitive pollutants

ContaminantSource

ZVI Barrier

AnaerobicPolishingZone

AerobicPolishingZone

Chemical reductionChemical reduction&&

BiodegradationBiodegradation

Bioaugmentation

Any Questions?

Any Questions?

Goya, The Shootings of May Third 1808

PFOS(perfluronated

octosulfonates)

S

O

O

O-C8F17

NO2O2N

O

NO2O2N

O

Musk Ketone(Synthetic musk fragance)

x + y = 1 to 10

O

BrxBry

O

BrxBry

Polybrominated Diphenyl Ethers(207 PBDE congeners)

Endocrine Disruptors Oxidized Rocket Propellants and Energetics

NO N

CH3

CH3

NDMA(N-Nitrisodimethylamine)

Perchlorate(ClO4

-)

O

O ClO

O

-

N

N

NNO2

NO2

O 2N

RDXHexahydro-1,3,5-trinitro-1,3,5-triazine

Emerging Pollutants

Cr(VI) Inhibited Abiotic CCl4 Degradation at Low but not at High Fe(0) Area Concentration

Time (Hours)

0 2 4 6 8 10

0

200

400

600

800

1000

1200

Time (Hours)

0 20 40 60 80 100 120 140 160

0

200

400

600

800

1000

1200

1400

High Fe(0) dose (1140 m2/L) Low Fe(0) dose (11 m2/L)

CC

l 4 (

g/L

)

CC

l 4 (

g/L

)

Alone AloneWith Cr(VI)

With Cr(VI)

Preferential Degradation: Cr(VI) > CCl4 > NO3- >>>>>>SO4

-2

Fernandez-Sanchez, Sawvel, and Alvarez (2003). Chemosphere. 54 (7): 923-829.

Less NO2- Eluted Initially from Bioaugmented Column

2 d 1 d 0.5 d 0.25 d

1 d

HRT

Time, days

0 20 40 60 80 100 120 140

Effl

uent

Nitr

ite C

onc.

, mg/

L-N

0

2

4

6

8

Fe(0)

Fe(0) + cellsMCL

Dejournett T. and P.J.J. Alvarez (2000). Bioremediation Journal 4:149-154.