the basic science of anaerobic bioremediation - · pdf filehydrogenolysis hydrogenation...

The basic science

of anaerobic bioremediation

Dan Leigh PG, CHG

June 4, 2013

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Introduction: Dan Leigh

– Licensed geologist and hydrogeologist

– Walnut Creek, CA

– Applying bioremediation for > 25 yrs

– Applying anaerobic bioremediation of chlorinated

organics for >20 yrs

– Currently working on development of

biogeochemical processes occurring during

anaerobic bioremediation

– [email protected]

– 925.984.9121

2 Basic Science of Anaerobic Bioremediation


mailto:[email protected]

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Presentation outline

• Basic concepts of biological and geochemical processes

– Respiration, fermentation, co metabolism

– Electron donors and acceptors

– Biotic and abiotic anaerobic degradation pathways of chlorinated

ethenes

– Processes for stimulating anaerobic bioremediation of chlorinated

organics

• Significant site conditions not conducive to anaerobic

bioremedation and how to overcome them

– Inappropriate or insufficient bacteria

– High dissolved oxygen

– Low pH

– High sulfate concentrations

• Biogeochemical degradation

• Summary



Contaminants that can be degraded

by anaerobic processes • Chlorinated solvents such as PCE, TCE, TCA, DCA,

CCl4, chloroform and methylene chloride

• Chlorobenzenes including di- and tri-chlorobenzene

• Energetic compounds such as TNT, DNT, HMX, RDX,

nitroglycerine and perchlorate.

• Most pesticides including DDT, DDE, dieldrin, 2,4-D and

2,4,5-T

• Nitrate compounds

• Petroleum hydrocarbons

This presentation focuses on biological and

geochemical processes that occur during the in situ

anaerobic degradation of chlorinated ethenes.



Bioremediation is a natural and

sustainable remediation process.

Bioremediation utilizes the life processes of

organisms to reduce the concentration,

mass, mobility or toxicity of contaminants.

– Yeast, fungi, bacteria or plants are

stimulated to degrade toxic substances.

– The primary processes include

respiration and fermentation.

– Not a new technology –

• e.g. wastewater treatment

– Improvements to bioremediation

approaches are being developed.



Basic concepts of biological and

geochemical processes

• Several biological processes occur during anaerobic

bioremediation including: – Respiration: Aerobic and Anaerobic

– Fermentation

– Co-metabolism

• Abiotic processes can be integrated, or occur naturally,

which enhance biological degradation processes.

• Biotic and abiotic anaerobic degradation processes

occur in distinct, identifiable pathways.



Respiration processes

Aerobic

Respiration

Aerobic

Respiration

Eating and breathing Electron

Donor

Electron

Acceptor Organism Respiration



Aerobic and anaerobic respiration

• Aerobic respiration

– Molecular oxygen (O2) is the only

electron acceptor used in the process

• Anaerobic respiration

– Any inorganic electron acceptor (other

than oxygen) is used in the respiration

process

• NO3, Mn(IV), As(V), Fe(III), SO4, CO2

• Cr(VI), ClO4



Respiration Biologically Mediated Oxidation - Reduction

Electron Donor Electron Acceptor

Resistor

Positive Negative

Growth Protein Synthesis

Reproduction

CnHn Fe(II)

H2S

H2

O2

NO3

As(V)

Mn(IV)

SO4

CO2

Work Light bulb

Motors

As(III)

Mn(II)

Fe (III) Reduced Oxidized

HNO2



Oxygen O2 + 4H+ + 4e- 2H2O (Eh0 = +820)

Nitrate 2NO3- + 12H+ +10e- N2(g) + 6H2O (Eh0 = +740)

De

cre

asin

g A

mo

un

t o

f En

erg

y R

ele

ase

d D

uri

ng

Elec

tro

n T

ran

sfe

r

Manganese (IV) MnO2(s) + HCO3 +3H + + 2e - MnCO3 (s) + 2H20 (Eh0 = +520)

Iron FeOOH(s) +HCO3 - + 2H+ e- FeCO3 + 2H2O (Eh0 = -50)

500

Aerobic

Anaerobic

1000

0

-250

Arsenic (V) H3AsO4 + 2H+ +2e- H3AsO3 + H2O (Eh0 = +559)

Chromium (VI ) Cr2O72- + 14H+ + 6e- 2Cr3++7H2O (Eh0 = +1330)

Anaerobic

Eh range for various electron acceptors

Redox Potential (Eh0) in Millivolts @ pH = 7

and T = 250C

Methanogenesis CO2 + 8H+ + 8e- CH4 + 2H2O (Eh0 = -240)

Sulfate SO4 2- + 9H+ + 8e- HS- + 4H2O (Eh0 = -220)



Anaerobic respiration and chlororespiration

Anaerobic

Respiration

Chlororespiration

Electron

Donor

Electron

Acceptor Biota Respiration

Aerobic

Respiration

NO3

SO4

Fe(III)

CO2

Mn(IV)



Range for Effective Chlorinated Ethene

Degradation (chlororespiration)

↓

Methanogenesis CO2 + 8H+ + 8e- CH4 + 2H2O (Eh0 = -240)

Sulfate SO4 2- + 9H+ + 8e- HS- + 4H2O (Eh0 = -220)

Iron FeOOH(s) +HCO3 - + 2H+ e- FeCO3 + 2H2O (Eh0 = -50)

Oxygen O2 + 4H+ + 4e- 2H2O (Eh0 = +820)

Nitrate 2NO3- + 12H+ +10e- N2(g) + 6H2O (Eh0 = +740)

De

cre

asin

g A

mo

un

t o

f En

erg

y R

ele

ase

d D

uri

ng

Elec

tro

n T

ran

sfe

r

Manganese (IV) MnO2(s) + HCO3 +3H + + 2e - MnCO3 (s) + 2H20 (Eh0 = +520)

Redox Potential (Eh0) in Millivolts @ pH = 7

and T = 250C

500

Aerobic

Anaerobic

1000

0

-250

Arsenic (V) H3AsO4 + 2H+ +2e- H3AsO3 + H2O (Eh0 = +559)

Chromium (VI ) Cr2O72- + 14H+ + 6e- 2Cr3++7H2O (Eh0 = +1330)

Anaerobic

Eh range for cholorinated ethene degradation

PCE TCE

TCE DCE

DCE VC

VC Ethene



Many organisms generate energy by

fermentation rather than respiration • Fermentation refers to the conversion of sugar to acids,

gases and/or alcohol using yeast or bacteria.

• Fermentation does not use an electron transport chain

(e.g. O2, NO3, Mn(IV), SO4, CO2) as does respiration.

• Fermentation uses a reduced carbon source (e.g.,

cellulose, lecithin, lactose, sugars).

– to generate volatile fatty acids ((VFAs) e.g. lactic, acetic,

propionic, valeric, butyric acids)

– and gases (e.g. H2, CO2, CH4)

• H2 is used by dechlorinating bacteria to generate

energy by sequentially reducing chlorinated organics.



A note about

co-metabolic oxidation The microbial breakdown of a contaminant in which the contaminant is

oxidized incidentally by an enzyme or cofactor that is produced during

microbial metabolism of another compound is called aerobic/anaerobic

co-metabolism.

– Co-metabolic oxidation applies respiration processes:

• Electron donor: (e.g., methane, ethane, ethene, propane, butane, toluene, phenol,

ammonia) PLUS: electron acceptor (e.g, O2, SO4)

– Enzymes generated to degrade food source also fortuitously degrades CEs or

other contaminants.

– The degrading organism does not gain energy from the contaminant degradation.

– The presence of electron donor may inhibit contaminant degradation.

Co-metabolism can be a challenge to apply.

– Often requires substantial engineering effort

– It is difficult to identify co-metabolic degradation in the aquifer

– May not be an efficient use of substrate



Dechlorinating bacteria

• Several organisms capable of

partially dechlorinating

chlorinated organics.

• Only organism confirmed to

dechlorinate DCE and VC to

ethene is Dehalococcoides

(Dhc).

• Dhc uses H2 as the electron

donor in dechlorination process.



Biological Reductive Dechlorination of Chlorinated Ethenes

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

H H H H H H

PCE PCE PCE TCE TCE TCE cis 1,2 -DCE trans 1,2 -DCE 1,1 -DCE

H H

H H H H H H H H H H

VC VC VC

H H

H H H H

Ethene Ethene Ethene

0

- 50

- 200

- 150

- 250

ORP


C C C C C C


β elimination (abiotic) pathway

Cl

C

Cl

Cl

Cl Cl Cl

H

Cl

Cl

Cl

PCE TCE Dichloroacetylene Chloroacetylene Acetylene DCE

H H

Fe 0

II

Fe 0

II

Fe 0

II

C C C C C

Ethene Ethane


Hydrogenation Hydrogenolysis

Acetylene


Some Hypothesized Reaction Pathways

Biotic Abiotic PCE

TCE

Cis 1,2-DCE Trans 1,2-DCE

VC

Ethene

Ethane

PCE

TCE

VC

Ethene

Ethane

Chloroacetylene

Acetylene

1,1-DCE, trans 1,2-DCE, cis1,2-DCE

Dichloroacetylene

Hydrogenolysis

β-elimination

α-elimination

Hydrogenation

CO2 , CH4 , H2O

CO2, CH4,H2O



Concentr

ation

Time

Concentr

ation

Time

Biological and abiotic degradation processes appear

different when measuring standard analytical parameters

Biological Degradation

(Chlororespiration) Abiotic Degradation

PCE TCE DCE VC Ethene

(β elimination)

Anticipated change in CE molar concentration

Total



Generating anaerobic

bioremediation processes Enhanced anaerobic bioremediation is conducted by providing

whatever is limiting the complete degradation process.

Need appropriate organism and electron donor (H2) to degrade CEs

Other supplements can be made to further enhance the anaerobic

process.

– Chemical reductants (e.g. ZVI, ferrous iron)

– Nutrients

Additional supplements can be made to enhance synergistic effects.

– Sulfate

– Iron


Electron

Donor

Electron

Acceptor Organism Chlororespiration


Molasses

Starch

Cheese whey

Emulsified vegetable oil

Corn syrup

Lactose

Glucose

Ethanol

Methanol

Propanol

Lecithin

Glycerol, xylitol, sorbitol

Polylactate esters of fatty acids (e.g.., Glycerol tripolylactate)

Acetic acid and its salts

Lactic acid and its salts

Propionic acid and its salts

Citric acid and its salts

Benzoic acid and its salts

Oleic acid and its salts

Various Bean Oils (soy, guar)

Complex sugars

Food process byproducts including milk whey or yeast extract

Complex organic material such as wood chips (cellulose)

Draft General Waste Discharge

Requirements for

In Situ Groundwater Remediation –

Santa Ana Water Quality Control

Board CA, 2013

Only H2 has been

shown to be an

electron donor for

cis 1,2-DCE and

vinyl chloride

conversion to

ethene

Anaerobic reductive dechlorination is stimulated by

providing an electron donor to the organisms

Molecular Hydrogen (H2)

Various substrates used to generate H2 for dechlorination:



Electron Acceptor Electron equivalents per mole

Oxygen (dissolved) 4

Nitrate (dissolved) 4

Sulfate (dissolved/solid) 8

Maybe carbon dioxide (dissolved) 8

Manganese (IV) (solid) 2

Ferric iron (III) (Solid) 1

PCE – tetrachloroethene (dissolved + adsorbed + NAPL) 8

TCE – trichloroethene (dissolved adsorbed + NAPL) 6

DCE – dichloroethene (dissolved + adsorbed) 4

VC – vinyl chloride (dissolved + adsorbed) 2

Most of the contaminant mass may be adsorbed to

aquifer matrix

Substrate requirements partially determined by amount

of hydrogen required to reduce electron acceptors and

contaminants



Some electron acceptors

may be in solid form

• Solid electron acceptors

occur as: • oxides

• salts

• minerals

• Solid electron

acceptors are not

accounted for by

dissolved phase

analysis.

Some mineral electron

acceptors

• Barite – BaSO4

• Gypsum – CaSO4·2H2O

• Anhydrite – CaSO4

• Hannebachite – CaSO3 ·0.5H2O

• Anglesite (PbSO4)

• Magnetite (Fe2+Fe3+2O4 or Fe3O4)

• Hematite (Fe2O3)

Barite

(BaSO4)



Substrate requirements partially determined by amount

of hydrogen generated during fermentation

Electron Donor Electron equivalent per mole

acetate 4

proprionate 3

lactate 2

fructose/glucose 12

sucrose/lactose 24

cellulose 24

linoleic acid 50

glycerol 7

lecithin 122 Most data derived from Fennel & Gossett (1998) and He, et al (2002)

Hydrogen equivalents produced by various electron donors



Draft General Waste Discharge

Requirements for

In Situ Groundwater Remediation – Santa

Ana Water Quality Control Board CA, 2013

Ferrous Chloride

Ferrous Carbonate

Ferrous Gluconate

Sorbitol Cysteinate

Sodium Sulfide

Sodium Dithionite

Calcium Polysulfide

Zero-Valent Iron

Granular

Emulsified

Micro-scale

Nano-scale

Reducing/reductive degradation

enhancement compounds



Undesired and unexpected results

Incomplete degradation (e.g. cis DCE or VC stall)

• No, or insufficient Dhc population

• Insufficient /too much substrate

• Inefficient distribution of substrate and culture

• Geochemical issues (e.g., sulfide toxicity)

• pH outside appropriate range

Contaminants disappear without generation of daughter products

• May be partitioning into substrate

• May be biogeochemical/abiotic degradation

Contaminants disappear but come back after substrate is gone. • Other source of contaminants

• DNAPL possible

• High adsorbed phase

• Matrix diffusion



Anaerobic bioremediation may be applicable at

more sites than previously considered.

Some sites may not initially appear to be

appropriate for anaerobic bioremediation. Some of

these conditions include: • Inappropriate or insufficient dechlorinating bacteria

• High dissolved oxygen concentration

• Low pH

• Very high sulfate concentrations

Modifications may be made to alleviate these

conditions and allow use of anaerobic

bioremediation.



At some sites biostimulation is sufficient, at

other sites bioaugmentation is required.

• Biostimulation is the

modification of the

environment to stimulate

existing bacteria capable

of bioremediation.

– Nutrients – e.g. nitrogen,

phosphorous, potassium

– Electron acceptors – e.g.

oxygen, nitrate,

manganese, ferric iron,

sulfate carbon dioxide

– Electron donors – e.g.

lactate, vegetable oil,

lecithin, cellulose, lactose

• Bioaugmentation is the

introduction of a group of

natural microbial strains

or genetically engineered

variants to achieve

bioremediation.

– Indigenous – native to site

– Exogenous - introduced



Is bioaugmentation necessary?

• Dechlorinating organisms may not be present at

sufficient concentrations at many sites.

– > 1x107 Dhc cells/L considered necessary for dechlorination

• The indigenous organism may not be efficient at

dechlorination.

– Final step may be co-metabolic, which is slow

• Indigenous organisms (e.g. methanogenic bacteria) may

outcompete dechlorinators such as (Dhc) for H2.

www.mdsg.umd.edu/CQ/v05n1/main/



Various organisms

approved for bioaugmentation

Dehalococcoides (Dhc)

Dehalobacter

Dehalogenimonas

Desulfuromonas

Desulfitobacterium

Desulfovbrio

Sulfurospirillum

Alcaligenes faecalis

Arthrobacter

Geobacter

Corynebacterium

Nitrosomonas

Nitrobacter

Rhodococcus

Pseudomonas fluorescens

Methylibium petroleiphilum

Methanotrophs

Methylosinus



ETHENES LOOP 3 (BIOSTIMULATION, LACTATE ONLY)

0

50

100

150

200

0 30 60 90 120 150 180 210 240 270 300 330 360

Days

Co

nc

en

tra

tio

n (m

mo

l/L

)

Tetrachloroethene

Trichloroethene

1,2-Dichloroethene (total)

Vinyl Chloride

Ethene

Total umol/L

Biostimulation only

Bioaugmentation can increase degradation rates



ETHENES LOOP 2 (BIOAUGMENTATION, LACTATE )

0

50

100

150

200

250

300

350

400

0 30 60 90 120 150 180 210 240 270 300 330 360

Days

Co

ncen

trati

on

(mm

ol/L

)

Tetrachloroethene

Trichloroethene

1,2-Dichloroethene (total)

Vinyl Chloride

Ethene

Total umol/L

Comparison of bioaugmentation to biostimulation

Biostimulation with Bioaugmentation

High total molar concentration



Can anaerobic processes be applied in

aerobic aquifers?

• Aerobic aquifers are often not considered appropriate for

the application of anaerobic biological processes.

• Bioaugmentation is necessary to treat CE’s biologically in

aerobic aquifers.

• Substantial effort is considered necessary to bioaugment

in aerobic aquifers (i.e., several injection events required

to establish reducing conditions).

– Suggests anaerobic bio treatment not cost effective.



Plan View

Cross Section

Inject 25% Substrate Inject Anaerobic Chase Water Inject Bioaugmentation Culture Inject Chase Water Inject 75% Substrate

Bioaugmentation methods applied to

overcome aerobic conditions



Sites with high dissolved oxygen can be

appropriate for anaerobic bioremediation

• Dhc is an obligate anaerobe

– Anaerobes are organisms that are not able to use (consume)

molecular oxygen.

– Obligate: those that cannot grow in the presence of molecular

oxygen.

• Anaerobic bacteria can be:

– Oxyduric: those that are not killed by (i.e. tolerant of) molecular

oxygen.

– Oxylabile: Those killed in the presence of molecular oxygen.

– Aerotolerant: those able to grow in the presence of molecular

oxygen even though they do not use it.



Bioaugmentation methods applied to

overcome aerobic conditions

Dhc exposed to oxygen in GW


DO depletion in closed system after

addition of SDC-9* and e- donor

Time (minutes)

2

4

5

6

7

DO

Co

ncen

trati

on

(m

g/L

)

3

100 200 300 400 500 0 1

Temperature 15 ± °C

TSS 0.1 g/L

DHC Concentration 9E10 cells/L

*SDC-9 is a trademark of the CB&I/Shaw Corporation



cDCE and VC degradation rates by SDC-9

exposed to air (with & without e- donor)

10 20 30 40 50 60 70 80 0 0

5

10

15

20

25

Air Exposure Time (Hours)

Deg

rad

ati

on

Rate

(m

g/L

xh

)

VC - Air Exposure

cDCE - Air Exposure

VC – e- donor - Air Exposure

cDCE – e- donor - Air Exposure

cDCE - Anaerobic Control No Air Exposure

VC - Anaerobic Control No Air Exposure

DHC 5E10 copies/L

Temperature 15±°C

Leigh, D.P., S. Vainberg, and R.Steffan, R., 2013, Can

Anaerobic Bioaugmentation Cultures be Applied Directly to

Aerobic Aquifers?: In situ and on Site Bioremediation

Symposium, 2013.



0

1

2

3

4

5

6

7

8

-100 -50 0 50 100 150 200 250 300

mg

/L

Days (Day 0 = June 6, 2011)

CNWS - Dissolved Oxygen

Field analytical results

Dissolved Oxygen



Groundwater analytical results after

bioaugmentation of anaerobic culture into an

aerobic aquifer

0

1

10

100

1000

10000

-100 0 100 200 300 400 500

µg

/L


Trichloroethene (TCE)

0

200

400

600

800

1000

1200

-100 0 100 200 300 400 500

Co

nc

en

tra

tio

n (µ

g/L

)


Total Dichloroethene (DCE)

0

1

10

100

1000

-100 0 100 200 300 400 500

Co

ncen

trati

on

( µ

g/L

)


Vinyl Chloride (VC)

0

20

40

60

80

100

120

-100 0 100 200 300 400 500

Co

nc

en

tra

tio

n( µ

g/L

)


Ethene



Anerobic biodegradation can be

conducted only in a defined range of pH

• Dhc species are very sensitive to pH.

• Some other organisms (e.g.

methanogens/SRBs) are not as sensitive to

pH.

• SRB’s and methanogens outcompete

dechlorinators for available H2.

• Addition of organic substrates can generate

organic acids which cause pH drop.

• Addition of ZVI/buffers raises pH. 42 Basic Science of Anaerobic Bioremediation


Dechlorination rates by Dhc are affected by pH

6 5 10 7 8 9 pH

0

0.5

1.0

1.5

Vainberg, S., C.W. Condee, R.J. Steffan. 2009. Large scale production of Dehalococcoides sp.-

containing cultures for bioaugmentation. J. Indust. Microbiol. Biotechnol. 36:1189-1197.


Dhc do not recover the

ability to dechlorinate after

extended exposure to low

pH water.


Elevated concentrations of sulfide can

inhibit anaerobic biodegradation

• Sulfate reduction stimulated

during anaerobic bioremediation

• Sulfate converted into HS-

• If ferrous iron is present, it will

precipitate as ferrous sulfide

species such as pyrite and

mackinawite

• If iron is insufficient, toxic levels

of HS- may accumulate.

Addition of iron can solve sulfide

toxicity issues.



Example of sulfide toxicity

Bench tests – ambient conditions

Time (weeks)

1000

100

10

1

0.1

0.01

0.001

Concentr

ation (

mg/L

)

Sulfate

& S

ulfid

e C

oncentr

ation (

mg/L

)

1200

1000

800

600

400

200

0

0 4 8 12 16 20 24 28 32

e- donor

Addition

Week 8

Bioaugmentation Week 17

e- donor

Addition

Week 20

TCE DCE VC Ethene Sulfate Sulfide



Time (weeks)

1000

100

10

1

0.1

0.01

0.001

Concentr

ation (

mg/L

)

Su

lfa

te &

Su

lfid

e C

on

cen

tra

tio

n (

mg

/L) 1200

1000

800

600

400

200

0

0 4 8 12 16 20 24 28 32

e- donor

Addition

Week 8

Bioaugmentation Week 17

e- donor

Addition

Week 20

TCE DCE VC Ethene Sulfate Sulfide

Example of sulfide toxicity

Bench tests – Fe-sulfide precipitation



Anaerobic biogeochemical degradation

• Reactive iron sulfide minerals are produced

at sites containing bioavailble iron and

sulfate during anaerobic bioremediation.

• Degradation occurs by contact with reactive

minerals

• Biogeochemical degradation pathway are

the same as for ZVI (β elimination).

Biogeochemical degradation includes processes where

contaminants are degraded by abiotic reactions with naturally

occurring and biogenically-formed minerals in the

subsurface.



Reactive iron sulfides minerals are formed during

anaerobic bioremediation processes

Pyrite (FeS2) Mackinawite (Fe(1+x)S

Euhedral pyrite (FeS2) Mackinawite (FeS)

pore coatings

Framboidal

Pyrite

(FeS2)

Mackinawite

coating

Pyrite

Framboids



Other potential applications of

anaerobic bioremediation

• Sequential anaerobic/aerobic bioremediation can be applied

to treat some contaminants (i.e, chlorobenzenes/CEs).

• Sulfate generated during activated persulfate treatment can

be reduced to generate reactive iron sulfides.

• Biogeochemical processes occuring with anaerobic

bioremediation can be enhanced to sequester metals.

• Enhanced anaerobic bioremediation can be applied following

thermal treatment.

• Anaerobic bioremediation can be applied to supplement or

replace existing pump and treat systems.



Presentation Summary

• Bioremediation uses natural and sustainable processes to

destroy contaminants rather than transfer to other media.

• The bioremediation process is effective because it enhances

the life processes of the organisms.

• Because this technology uses life processes organisms it can

be applied at sites with very high contaminant concentrations.

• Anaerobic bioremediation can be enhanced by adding abiotic

substrates (ZVI, soluble iron) and biogeochemical

amendments (sulfur sources) depending on site conditions.

• Anaerobic bioremediation can be conducted in aquifers

exhibiting low pH, high DO or high sulfate concentrations.

• Combined anaerobic biological, abiotic and biogeochemical

processes effectively treats a wide range of contaminants in

soil and groundwater.




[email protected]

925.984.9121


mailto:[email protected]

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