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Hydrogen Enhancement of Sediment Hydrogen Enhancement of Sediment Microbial Activity and Microbial Activity and
Contaminant DegradationContaminant Degradation
Peter Adriaens, Ph.D.Peter Adriaens, Ph.D.Civil and Environmental EngineeringCivil and Environmental Engineering
University of MichiganUniversity of Michigan
John Wolfe, Ph.D.John Wolfe, Ph.D.LimnoTechLimnoTech, Inc., Inc.
ETC Workshop on ETC Workshop on In SituIn Situ Technology Benchmarking for Technology Benchmarking for Sediment and Floodplain RemediationSediment and Floodplain Remediation
Ann Arbor, MIAnn Arbor, MIMarch 25March 25--26, 200426, 2004
The Technology
HypothesisRationaleApproach and MethodsHydrogen-Impacted Microbial EcologyHydrogen-Enhanced DechlorinationScientific ChallengesBench-Scale Technology DevelopmentTechnological Challenges
Hypotheses for Hydrogen-Based Enhancement
In situ amendment with hydrogen can increase metabolic and dechlorinationactivityThe technology is scalableThe technology can be cost-effectively applied to large and complex contaminated areas
Rationale for Hydrogen-Based Technologies
Ambient carbon and hydrogen fluxes limit in situ microbial activity in reducing soils and sediments– 5-20% of total extractable populationIncreased hydrogen fluxes enhance total respiratory competence and influence ecological composition– 15-80% of total extractable populationHydrogen gas is cheap and diffuses rapidly in sediments
Fundamental Process Understanding: Evidence of Dioxin Dechlorination
Electron acceptors:DOM
Electron acceptors:Bicarbonate
Sulfate
Electron acceptors:Bicarbonate,
Natural (river bottom water)
Electron donors:Sulfide
Ti-citrateSediment microorganisms
Electron donors/primers:Organic acids(Hydrogen)
2-MonobromoDD
Electron donors/primers:Organic acids
Hydrogen2-MonobromoDD
Dioxin Source:Estuarine-spiked HpCDD (both
isomers)
Dioxin Source:Freshwater-spiked OCDDFreshwater-hist. residuesEstuarine-spiked HpCDD
(both isomers separately)Marine-spiked HpCDD
Dioxin Source:Freshwater-spiked Penta- to
octaCDDEstuarine-historical residues
Monomers:Catechol, resorcinol, 3,4-
dihydroxybenzoic acidPolymers:
Polymaleic acid, Aldrich humic
Sediment-eluted mixed communities
Passaic River coresHudson River core
Model DOMMicroorganismsSediments
O
O
ClCl
ClCl
Cl
Cl
Cl
Cl
O
O
Cl
ClCl
Cl
Cl
Cl
Cl
O
O
C l
ClCl
Cl
Cl
Cl
Cl
O
O
Cl
ClCl
ClCl
Cl
O
O
Cl
Cl
Cl
Cl
Cl
Cl
O
O
Cl
Cl
Cl
Cl
O
O
C l
Cl
Cl
Cl
Cl
Cl
O
O
C l
ClCl
Cl
Cl
Cl
O
OClCl
Cl
Cl
Cl
Cl
O
O
Cl
O
O
Cl
1,2,3,4,6,7,8-heptaCDD
1,2,3,4,6,7,9-heptaCDD
1,2,3,4,6,7,8,9-octaCDD
2,3,7,8-tetraCDD
1,2,3,6,7,8-hexaCDD
1,2,3,4,7,8-hexaCDD
+ 1,2,3,7,8pentaCDD
Non-2,3,7,8tetraCDD
1,2,4,6,7,9-hexaCDD
+
1,2,3,4,6,8-hexaCDD
1,2,3,4,6,7-hexaCDD
Non-2,3,7,8pentaCDD
2-monoCDD
1-monoCDD
diCDDtriCDD1
2
6
89
34
7
Peri-dechlorination pathway
Peri-lateral dechlorination pathway
Differentiation of peri (1469)- and lateral (2378)- Dechlorination Pathways
Influence of Sediment Geochemistry on Dioxin Reactivity
Nutrient-rich OligotrophicIncreasing salinity/sulfate concentrations
Freshwater Estuarine Marine
CH4-H2 CH4-H2 /HS- CH4/HS- HS-
CH2O
H2
Acetate CO2 CO2
CH2O CH2O
H2 H2
PCDD PCDD PCDD
Peri < lateral peri < lateral peri << lateral
BiogeochemicalDesignation
Dominantdechlorination
pathway
Dominantcarbon flow
DominantTEAP
Impact of Hydrogen on Microbial PCDD Dechlorination in Sediments
0
10
20
30
40
50
60
70
80
1 2 3 4 5
Treatment
OCDD 1,2,3,4,6,7,8-HpCDD 1,2,3,4,6,7,9-HpCDD2,3,7,8-TCDD 2-MCDD
Original Hydrogen Acids2378-TCDD (mol%): 20 12 20Endpoint: tetra mono tetraRate (pmol TCDD/day): NA 28.6 -0.4 (net formation)
Unamended Heat-treated Organic acids BrDD primer Hydrogen
Hydrogen Technology Scaling: Laboratory Studies (EPA-SITE program)
Matrix:– Cell elution– Slurry– Column
Treatment: H2 addition, HCB spike
Response:– Microbial activity– Contaminant degradation
Process-levellab study
Fieldapplication
ScaleSystem complexity
--
++
Experimental Matrix (marine harbor sediment)
Cell elution Sediment slurry Sediment column
Sediment core
Methods: Hydrogen Enhancement of Elutions and Slurries
Sediment-eluted microorganisms are dispensed in the SIXFORSsystem in sulfate-rich estuarine media.
The reactors are amended with varying H2fluxes to prime cells.
– Sparged with H2/N2 mix including up to 1% H2
Organic acid cocktail added at t=0: 10 mg/L benzoic + 15 mg/L butyric + 75 mg/L acetic
This 6-reactor system is equipped with a H2/N2 gas
mixing/delivery system, temperature, and pH control.
Microbial Metabolic Response to Hydrogen:Redox dye (CTC) measurements
cell wallcell wall
Active oxidativeActive oxidativeenzymes (respiration)enzymes (respiration)
DissolvedDissolvedredox dyeredox dye(colorless)(colorless)
Reduced Redox DyeReduced Redox Dye(colored PPT)(colored PPT)
ALIVEALIVEMetabolically Competent Metabolically Competent INACTIVE / DEADINACTIVE / DEAD
+
+
Must be used in conjunction with cell wall stains
CTC (bright red)CTC (bright red)
cell wallcell wall
Active oxidativeActive oxidativeenzymes (respiration)enzymes (respiration)
DissolvedDissolvedredox dyeredox dye(colorless)(colorless)
Reduced Redox DyeReduced Redox Dye(colored PPT)(colored PPT)
ALIVEALIVEMetabolically Competent Metabolically Competent INACTIVE / DEADINACTIVE / DEADINACTIVE / DEADINACTIVE / DEAD
+
+
Must be used in conjunction with cell wall stains
CTC (bright red)CTC (bright red)
Microscope analysis:– Green – nonactive cell– Green/red – active cell
Flow Cytometry: Cell number and activity quantification
Automates cell counting– Density with green
fluorescence (FL1) gives total cells
– Density with red fluorescence (FL3) gives active cells
– About 5% of cells typically CTC active (Marine Harbor sample)
R1
R2
R1
R3R3
R1
Ecological Response to Hydrogen: Flow Cytometry Analysis (Passaic R.)• Microbial population density (measured using
PicoGreen): R1 = total eluted bacteria; R3 = bacteria present at elevated hydrogen concentrations (above CTC enhancement threshold)
• R3 represents less than 10% of total cell density, but is 80% CTC active
• Microbial community was analyzed using T-RFLP
Ecological Response to Hydrogen: T-RFLP Analysis (Passaic River)
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
background no H2below
thresholdabove
threshold
Rel
ativ
e ab
unda
nce
of T
-RFs
(%)
new "above threshold" T-RFsnew treatment vs. background T-RFsbackground T-RFs
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
background no H2below
thresholdabove
threshold
Rel
ativ
e ab
unda
nce
of T
-RFs
(%)
new "above threshold" T-RFsnew treatment vs. background T-RFsbackground T-RFs
Amendments of microbial elutions with nitrogen gas (no H2) and H2 fluxes not impacting CTC activity result in 20-30% emerging T-RFsAmendments above threshold of CTC activity result in emergence of 20% distinct RFsNo populations (out of a total of 74 T-RFs) could be identified using MspICross-referencing and multi-database search using three restriction enzymes is underway
Activity Enhancement for Three Sites –Based on Cell Elutions
1.0 – 3.5 µM H2 increases CTC activity ~ 3-fold
Activity Enhancement Experiments
0
5
10
15
20
25
- 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
H2 Concentration (µM)
% C
TC A
ctiv
ity
Marine Harbor Passaic San Diego
Baseline Activity Range (3-5%)
Activity Results - Slurry Study (Marine Harbor)
CTC activity increased about 3 fold
Cells counts increased about 8 fold
Pearl Harbor Slurry Study
0
2
4
6
8
10
12
14
16
18
Baseline Unenhanced Enhanced
% C
TC A
ctiv
e M
icro
orga
nism
s
Slurry Study
Pearl Harbor Slurry Study
1E+06
1E+07
1E+08
Baseline Unenhanced Enhanced
Mic
roor
gani
sms/
gram
dry
sed
imen
t
Slurry Study
H2 Amendment, Column Study (Marine Harbor)
Column Study: Top Water H2
05
1015
20
11/15 11/20 11/25 11/30 12/5 12/10 12/15 12/20
Caq
(nM
)Porewater H2 limited by diffusion– Leading edge advanced ~ 0.5’/month– Annual zone of influence up to ~6 feet
Activity Results - Column Study (Marine Harbor)
CTC activity increased about 4-fold in bottom layer
Cell counts increased about 3-fold in bottom layer
Pearl Harbor Column Study - Activity
0
5
10
15
20
25
Time-zero Top/Middle Bottom
% C
TC A
ctiv
e M
icro
orga
nism
s '
Results after 1 monthTime-zero
Column Study - Activity
Pearl Harbor Column Study - Cell Counts
1E+06
1E+07
1E+08
Time-zero Top/Middle Bottom
Mic
roor
gani
sms/
gram
dry
sed
imen
t '
Results after 1 monthTime-zero
Column Study – Cell Counts
HCB Results - Cell Elution Study (Marine Harbor)
H2 treatment increased HCB degradation rate by ~ 50%
Hydrogen Amendment
Degradation Rate (1/hr)
Initial HCB Concentration
(ppb)
HCB Concentration
after 48 hrs (ppb)
Change in HCB (%)
Below threshold
(< 0.5 µM H2)
0.0135 95% confidence interval:
0.0082 – 0.0188
5.6 (std dev 0.17)
3.2 (std dev 0.01)
43%
Above threshold
(0.6 µM H2)
0.0201 95% confidence interval:
0.0176 – 0.0225
8.7 (std dev 0.09)
3.2 (std dev 0.05)
63%
Above threshold
(1.8 µM H2)
0.0214 95% confidence interval:
0.0199 – 0.0228
6.8 (std dev 0.12)
2.5 (std dev 0.05)
63%
HCB Results: Slurry and Column Studies (Marine Harbor)
Slurry (at 2 months)– Treatment effects not
yet statistically significant
– Two future sampling events
Column (at 1 month)– Treatment effects not
yet statistically significant
– Two more columns
Pearl Harbor Slurry HCB Results
0.0
0.5
1.0
1.5
Time-zero 2 weeks 2 monthsHC
B M
ass
(M /
M 0)
, adj
for T
BB
reco
very
All Non-enhanced H2-amended
Slurry HCB Results
Column Study HCB Results
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Time-zero Top/Middle BottomHC
B C
onc
(C /
C 0)
, adj
for T
BB
reco
very
'
Results after 1 monthTime-zero
Column Study HCB Results
Scientific Challenges
Better understanding of hydrogen diffusion in sediment, including spatial distributionDevelopment of correlation between hydrogen enhancement, ecological response and dechlorination activityTemporal effect:– Amendment to CTC activity increase– CTC to dechlorination activity increase– Pulsed vs. continuous amendment– Limiting ratios of carbon to hydrogen– Impact of bioavailability on long term activity
Future Steps for Technology Development
Translate/scale effects on spiked HCB to effects on target contaminants
Key issues for introducing H2 in field:– As dissolved H2?– To what depth?– How to minimize resuspension? – Spacing of injection points?
What’s next?– Slurry and column studies to completion– Bench studies of H2 injection grid (H2-GRID) to
refine design parameters– Scale-up cost analysis– Design and conduct field pilot
Acknowledgements
Authors:– Peter Adriaens, Timothy Towey,
Nicole Dolney, University of Michigan– Cyndee Gruden, University of Toledo– John Wolfe, Richard McCulloch,
Limno-Tech, Inc.
Sponsors:– EPA/SITE– NOAA-CICEET
Disclaimer: Nothing in this presentation should be interpreted to imply that any product or service is endorsed or preferred by our research sponsors, or considered by those sponsors to be superior to other products or services.