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.