pedro j.j. alvarez, ph.d., p.e., dee iron-based bioremediation of aquifers contaminated with rdx or...
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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.