The basic science
of anaerobic bioremediation
Dan Leigh PG, CHG
June 4, 2013
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
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FMC provides a wide range of products for
application of anaerobic bioremediation,
biogeochemical and abiotic degradation
EHC®
Solid organic substrate with microscale ZVI
EHC-L ®
Liquid organic substrate with soluble Fe(II)
EHC-M ®
EHC® with sulfur source for biogeochemical
metals treatment
ELS ®
Emulsified Lecithin Substrate for
enhancement of anaerobic bioremediation
Daramend ®
Solid organic substrate with ZVI for
treatment of contaminated soils
http://environmental.fmc.com/solutions
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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
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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.
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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.
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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.
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Respiration processes
Eating and breathing
Organism
Electron
Donor
Electron
Acceptor
Respiration
Aerobic
Respiration
Aerobic
Respiration
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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
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Respiration Biologically Mediated Oxidation - Reduction
Growth
Protein Synthesis
Reproduction
Work
Light bulb
Motors
Resistor
Electron Donor
Negative
Electron Acceptor
Positive
Reduced
Oxidized
CnHn
HNO2
As(III)
Mn(II)
Fe(II)
H2S
H2
O2
Fe (III)
NO3
SO4
As(V)
CO2
Mn(IV)
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Decreasing Amount of Energy Released During Electron Transfer
Eh range for various electron acceptors
1000
Chromium (VI )
Cr2O72- + 14H+ + 6e-
2Cr3++7H2O (Eh0 = +1330)
Anaerobic
Aerobic
Anaerobic
500
Oxygen
Nitrate
N2(g) + 6H2O (Eh0 = +740)
2NO3- + 12H+ +10e-
Arsenic (V)
Manganese (IV)
H3AsO4 + 2H+ +2e-
H3AsO3 + H2O (Eh0 = +559)
MnCO3 (s) + 2H20
(Eh0 = +520)
MnO2(s) + HCO3 +3H + + 2e -
Redox Potential (Eh0)
in Millivolts @ pH = 7
and T = 250C
0
Iron
Sulfate
-250
2H2O (Eh0 = +820)
O2 + 4H+ + 4e-
FeOOH(s) +HCO3 - + 2H+ e-
SO4 2- + 9H+ + 8e-
Methanogenesis CO2 + 8H+ + 8e-
FeCO3 + 2H2O (Eh0 = -50)
HS- + 4H2O (Eh0 = -220)
CH4 + 2H2O (Eh0 = -240)
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Anaerobic respiration and chlororespiration
Biota
Electron
Donor
Electron
Acceptor
NO3
Mn(IV)
Fe(III)
SO4
CO2
Respiration
Aerobic
Anaerobic
Respiration
Respiration
Chlororespiration
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Decreasing Amount of Energy Released During Electron Transfer
Eh range for cholorinated ethene degradation
1000
Chromium (VI )
Cr2O72- + 14H+ + 6e-
2Cr3++7H2O (Eh0 = +1330)
Anaerobic
Aerobic
Anaerobic
500
Oxygen
Nitrate
Arsenic (V)
Manganese (IV)
Iron
H3AsO3 + H2O (Eh0 = +559)
MnCO3 (s) + 2H20
(Eh0 = +520)
MnO2(s) + HCO3 +3H + + 2e -
FeOOH(s) +HCO3 - + 2H+ e-
PCE
TCE
TCE
DCE
Sulfate
VC
-250
H3AsO4 + 2H+ +2e-
Redox Potential (Eh0)
in Millivolts @ pH = 7
and T = 250C
FeCO3 + 2H2O (Eh0 = -50)
VC
DCE
↓
N2(g) + 6H2O (Eh0 = +740)
2NO3- + 12H+ +10e-
0
Range for Effective
Chlorinated Ethene
Degradation
(chlororespiration)
2H2O (Eh0 = +820)
O2 + 4H+ + 4e-
2-
SO4 + 9H+ + 8e-
Ethene
Methanogenesis CO2 + 8H+ + 8e-
HS- + 4H2O (Eh0 = -220)
CH4 + 2H2O (Eh0 = -240)
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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.
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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
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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.
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Biological Reductive Dechlorination of Chlorinated Ethenes
ORP
0
- 50
Cl
HH
C
- 150
HH
Cl
Cl H
H
C
H
Cl H
H
H Cl
C
H
H Cl
Cl H
H
H ClH
C
C
H
H
Cl H H Cl
Cl H
H
C
H
Cl H
VC-DCE
Ethene
TCE
Ethene
VC
PCE
PCE
PCE
VC
cis Ethene
1,2
TCE
-DCE trans
TCE
1,2 -DCE 1,1
- 200
- 250
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β elimination (abiotic) pathway
Fe
0
Fe
0
Fe
0
Hydrogenation
Hydrogenolysis
II
Cl
Cl
C
Cl
II
H
C
C
Cl
Dichloroacetylene
PCE
Cl
II
Cl
H
C
H
C
Cl
Cl
Chloroacetylene
Acetylene
TCE
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Cl
DCE
Acetylene
Ethane
Ethene
18
Some Hypothesized Reaction Pathways
Biotic
Abiotic
PCE
TCE
PCE
Dichloroacetylene
TCE
Cis 1,2-DCE Trans 1,2-DCE
1,1-DCE, trans 1,2-DCE, cis1,2-DCE
VC
Chloroacetylene
VC
Ethene
Ethane
Acetylene
Ethene
Ethane
CO2 , CH4 , H2O
α-elimination
Hydrogenolysis
β-elimination
Hydrogenation
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CO2, CH4,H2O
Biological and abiotic degradation processes appear
different when measuring standard analytical parameters
Anticipated change in CE molar concentration
(Chlororespiration)
(β elimination)
Concentration
Abiotic Degradation
Concentration
Biological Degradation
Time
PCE
Time
TCE
DCE
VC
Total
Ethene
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Generating anaerobic
bioremediation processes
Enhanced anaerobic bioremediation is conducted by providing
whatever is limiting the complete degradation process.
Organism
Electron
Donor
Electron
Acceptor
Chlororespiration
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
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Anaerobic reductive dechlorination is stimulated by
providing an electron donor to the organisms
Various substrates used to generate H2 for dechlorination:
Molasses
Acetic acid and its salts
Starch
Lactic acid and its salts
Cheese whey
Propionic acid and its salts
Emulsified vegetable oil
Corn syrup
Citric acid and its salts
Various Bean Oils (soy, guar)
Lactose
Glucose
Ethanol
Benzoic acid and its salts
Only H2 has been
shown to be an
electron donor for
cis 1,2-DCE and
vinyl chloride
conversion to
ethene
Oleic acid and its salts
Methanol
Polylactate esters of fatty acids (e.g.., Glycerol tripolylactate)
Propanol
Food process byproducts including milk whey or yeast extract
Lecithin
Complex organic material such as wood chips (cellulose)
Glycerol, xylitol, sorbitol
Complex sugars
Molecular Hydrogen (H2)
Draft General Waste Discharge
Requirements for
In Situ Groundwater Remediation –
Santa Ana Water Quality Control
Board CA, 2013
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Substrate requirements partially determined by amount
of hydrogen required to reduce electron acceptors and
contaminants
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
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Some electron acceptors
may be in solid form
• Solid electron acceptors
occur as:
Some mineral electron
acceptors
Barite
(BaSO4)
• oxides
• salts
• minerals
• Solid electron
acceptors are not
accounted for by
dissolved phase
analysis.
•
•
•
•
•
•
•
Barite – BaSO4
Gypsum – CaSO4·2H2O
Anhydrite – CaSO4
Hannebachite – CaSO3 ·0.5H2O
Anglesite (PbSO4)
Magnetite (Fe2+Fe3+2O4 or Fe3O4)
Hematite (Fe2O3)
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Substrate requirements partially determined by amount
of hydrogen generated during fermentation
Hydrogen equivalents produced by various electron donors
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)
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Reducing/reductive degradation
enhancement compounds
Ferrous Chloride
Ferrous Carbonate
Ferrous Gluconate
Sorbitol Cysteinate
Sodium Dithionite
Calcium Polysulfide
Zero-Valent Iron
Granular
Emulsified
Draft General Waste Discharge
Requirements for
In Situ Groundwater Remediation – Santa
Ana Water Quality Control Board CA, 2013
Micro-scale
Sodium Sulfide
Nano-scale
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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
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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.
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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
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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/
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Various organisms
approved for bioaugmentation
Dehalococcoides (Dhc)
Geobacter
Dehalobacter
Corynebacterium
Dehalogenimonas
Nitrosomonas
Desulfuromonas
Nitrobacter
Desulfitobacterium
Rhodococcus
Desulfovbrio
Pseudomonas fluorescens
Sulfurospirillum
Methylibium petroleiphilum
Alcaligenes faecalis
Methanotrophs
Arthrobacter
Methylosinus
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Bioaugmentation can increase degradation rates
ETHENES LOOP
3 (BIOSTIMULATION,
LACTATE ONLY)
Biostimulation
only
200
Tetrachloroethene
Concentration (mmol/L)
Trichloroethene
1,2-Dichloroethene (total)
150
Vinyl Chloride
Ethene
Total umol/L
100
50
0
0
30
60
90
120
150
180
210
240
270
Days
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300
330
360
Comparison of bioaugmentation to biostimulation
ETHENES LOOP 2 (BIOAUGMENTATION,
LACTATE )
Biostimulation
with Bioaugmentation
400
Tetrachloroethene
Trichloroethene
Concentration (mmol/L)
350
1,2-Dichloroethene (total)
High total molar concentration
300
Vinyl Chloride
Ethene
250
Total umol/L
200
150
100
50
0
0
30
60
90
120
150
180
210
240
270
Days
Basic Science of Anaerobic Bioremediation
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300
330
360
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.
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Bioaugmentation methods applied to
overcome aerobic conditions
Plan View
Inject Anaerobic
25% Substrate
Bioaugmentation
Chase
75%
Water
Chase Culture
Water
Cross Section
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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.
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Bioaugmentation methods applied to
overcome aerobic conditions
Dhc exposed to oxygen in GW
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DO depletion in closed system after
addition of SDC-9* and e- donor
DO Concentration (mg/L)
7
Temperature 15 ± °C
TSS 0.1 g/L
DHC Concentration 9E10 cells/L
6
5
4
3
2
1
0
100
200
*SDC-9 is a trademark of the CB&I/Shaw Corporation
300
400
Time (minutes)
Basic Science of Anaerobic Bioremediation
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500
cDCE and VC degradation rates by SDC-9
exposed to air (with & without e- donor)
DHC 5E10 copies/L
Temperature 15±°C
25
Degradation Rate (mg/Lxh)
cDCE - Anaerobic Control No Air Exposure
VC - Anaerobic Control No Air Exposure
cDCE – e- donor - Air Exposure
20
VC – e- donor - Air Exposure
cDCE - Air Exposure
VC - Air Exposure
15
10
5
0
0
10
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.
20
30
40
50
60
Air Exposure Time (Hours)
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70
80
Field analytical results
Dissolved
Oxygen
CNWS - Dissolved
Oxygen
8
7
mg/L
6
5
4
3
2
1
0
-100
-50
0
50
100
150
200
250
Days (Day 0 = June 6, 2011)
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300
Groundwater analytical results after
bioaugmentation of anaerobic culture into an
aerobic aquifer
Total Dichloroethene (DCE)
Trichloroethene (TCE)
10000
Concentration (µg/L)
1200
µg/L
1000
100
10
1
0
-100
1000
800
600
400
200
0
100
200
300
400
0
-100
500
0
Days (Day 0 = June 6, 2011)
100
400
500
400
500
Ethene
120
Concentration( µg/L)
1000
Concentration( µg/L)
300
Days (Day 0 = June 6, 2011)
Vinyl Chloride (VC)
100
10
1
0
-100
200
0
100
200
300
400
500
100
80
60
40
20
0
-100
0
100
200
Days (Day 0 = June 6, 2011)
Days (Day 0 = June 6, 2011)
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300
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.
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Dechlorination rates by Dhc are affected by pH
1.5
1.0
Dhc do not recover the
ability to dechlorinate after
extended exposure to low
pH water.
0.5
0
5
6
7
8
9
pH
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.
Basic Science of Anaerobic Bioremediation
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10
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.
Basic Science of Anaerobic Bioremediation
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1200
Concentration (mg/L)
1000
Bioaugmentation Week 17
100
1000
10
800
1
600
0.1
400
e- donor
Addition
Week 8
0.01
e- donor
Addition
Week 20
200
0
0.001
0
TCE
4
DCE
8
VC
12
16
20
Time (weeks)
Ethene
Sulfate
24
28
Sulfide
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32
Sulfate & Sulfide Concentration (mg/L)
Example of sulfide toxicity
Bench tests – ambient conditions
1200
Concentration (mg/L)
1000
Bioaugmentation Week 17
100
1000
10
800
1
600
0.1
400
e- donor
Addition
Week 8
0.01
e- donor
Addition
Week 20
200
0
0.001
0
TCE
4
DCE
8
VC
12
16
20
Time (weeks)
Ethene
Sulfate
24
28
Sulfide
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32
Sulfate & Sulfide Concentration (mg/L)
Example of sulfide toxicity
Bench tests – Fe-sulfide precipitation
Anaerobic biogeochemical degradation
Biogeochemical degradation includes processes where
contaminants are degraded by abiotic reactions with naturally
occurring and biogenically-formed minerals in the
subsurface.
• 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).
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Reactive iron sulfides minerals are formed during
anaerobic bioremediation processes
Pyrite (FeS2)
Mackinawite (Fe(1+x)S
Mackinawite
coating
Pyrite
Framboids
Framboidal
Pyrite
(FeS2)
Euhedral pyrite (FeS2)
Mackinawite (FeS)
pore coatings
48
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.
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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.
Basic Science of Anaerobic Bioremediation
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Basic Science of Anaerobic Bioremediation
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