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Department of Chemical Engineering and Applied Chemistry
University of Toronto
Elizabeth A. Edwards
Anaerobic Benzene Biodegradation
Centre for Applied Bioscience and Bioengineering
Acknowledgements
Benzene Research team (past and current): Dr. FeiLuo, Shen Guo, Nancy Bawa, Elisse Magnuson, Johnny Xiao, Tommy Wang, Dr. Cheryl Devine, Dr. Roya Gitiafroz, Sarah McRae, Nancy Li, Julie Arrey, Dr. Ania Ulrich (now prof. at U of Alberta)
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Sandra Dworatzek, Jennifer Webb, Jennifer Wilkinson, and others
Kris Bradshaw, Rachel Peters
Colleagues: Krishna Mahadevan, Alexander Yakunin and Alexei Savchenko, Anna Kushnudinova, Veronica Yim, Linda Xu, Hong Cui
University of Freiburg – Enzyme Assays: Prof. Matthias Boll, Sebastian Estelmann, Dr. Brandon Morris
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Ward, D. M., Atlas, R. M., Boehm, P. D., & Calder, J. A. (1980). Microbial biodegradation and chemical evolution of oil from the Amoco spill. Ambio, 9(6), 277–283.
CH3
CH3 CH3
CH3
Benzene Toluene
Xylene(s)Ethylbenzene
3
1986
1987
1992
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Anaerobic Publications
0
5
10
15
20
25
30
35
40
80 86 87 88 89 90 91 92 93 94 95 96 97 98 99 00 01 02 03 04
Nu
mb
er o
f P
ub
licat
ion
s TEX Papers
B Papers
Presented at the ISME Conference2004 in Cancun
As of 2011:Identified 47 Anaerobic Benzene publications from 21
enrichments/cultures or sites… ever
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Contaminated Site Material(20 Years of Enrichment in the Lab)
Swamp
Sw-NO3
Cartwright
Cart-NO3 Cart-SO4 Cart-CH4
Oil Refinery (OR)
OR-CH4
Benzene-Degrading Enrichment Cultures
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Degrade: Benzene, Benzoate, Toluene, Phenol
Key Microbe: Peptococcaceae sp.
Degrade: BenzeneKey Microbe: δ-Proteobacterium “ORM2”
Nitrate-reducing Sulfate-reducing or methanogenic
Site 1 Site 2 Site 3
20 years
Known Anaerobic Benzene-DegradersArchaea
Betaproteobacteria
Firmicutes
Deltaproteobacteria
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Ferroglobus_placidus_NR_074531
Pepto_BpP102
Thermincola_NR_074717
Azoarcus_AN9_AB241406
Pelotomaculum_NR_040948
Pelotomaculum_NR_041320
Pepto_culture_BF
Pepto_Edlab
Geobacter_metallireducens
Azoarcus_NR_029266
Azoarcus_DN11_AB241403
Geobacter_Ben
Geobacter_daltonii
Desulfatitalea_NR_113315
Syntrophus_NR_102776
Desulfatiglans_NR_126176
HasdaA
ORM2
SB-21
BznS295
0.05
Iron (III)-reducing
Sulphate-reducing
Nitrate-reducing
Methanogenic
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Known Anaerobic Benzene-DegradersArchaea
Betaproteobacteria
Firmicutes
Deltaproteobacteria
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Ferroglobus_placidus_NR_074531
Pepto_BpP102
Thermincola_NR_074717
Azoarcus_AN9_AB241406
Pelotomaculum_NR_040948
Pelotomaculum_NR_041320
Pepto_culture_BF
Pepto_Edlab
Geobacter_metallireducens
Azoarcus_NR_029266
Azoarcus_DN11_AB241403
Geobacter_Ben
Geobacter_daltonii
Desulfatitalea_NR_113315
Syntrophus_NR_102776
Desulfatiglans_NR_126176
HasdaA
ORM2
SB-21
BznS295
0.05
Iron (III)-reducing
Sulphate-reducing
Nitrate-reducing
Methanogenic
Gulf of California, Mexico
Oil refinery, U.S.
Coal gasification site, Poland
Gasoline station, Canada
River sediment, Japan
Coal gasification site, Poland
Marine hydrothermal system, Italy
Petroleum contaminated site, Bemidji, U.S.Petroleum contaminated site, Bemidji, U.S.
Mediterranean lagoon, France
Gas contaminated aquifer, Japan
Gas contaminated aquifer, Japan
BTEX Degrading Enrichment Cultures: Rates
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Enrichment Culture
Substrates Main Organism Rate (mg/L/day)
Doubling Time
OR-CH4 Benzene/CO2 DeltaproteobacteriumORM20.3-2.0
20-30 daysCart-SO4 Benzene/SO4 DeltaproteobacteriumORM2
0.1-0.3
Sw-NO3 Benzene/NO3 Peptococcaceae sp. 0.1-0.4
Pen-CH4 Toluene and o-Xylene/CO2
Pelotomaculum sp. >10
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Questions I had posed in 2004• Why is it so difficult to obtain cultures capable of anaerobic
benzene degradation?Benzene Thermodynamically ~equivalent to Toluene
• Organisms/Processes don’t exist?• Organisms uncommon? (kinetic barrier?)• Inappropriate laboratory enrichment and culturing technique?
Where we are now in 2017?
B T
Questions I had posed in 2004• Why is it so difficult to obtain cultures capable of anaerobic
benzene degradation?Benzene Thermodynamically ~equivalent to Toluene
• Organisms/Processes don’t exist?• Organisms uncommon? (kinetic barrier?) YES; low abundance• Inappropriate laboratory enrichment and culturing technique? YES
Where we are now in 2017?
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In Search of Elusive Anaerobic Benzene Activation Mechanisms
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OH
CH3
COOH
COSCoA
HO
CH3 CO, CoA
CO2
?
Hydroxylation to phenol
Methylation to toluene
Carbonylation to benzaldehyde
Carboxylation to benzoate
Other????
bssA
ppsA, ppcA
bclABenzoyl-CoA is a central metaboliteabcA
1. Nitrate-Reducing Cultures
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BenzenePeptococcaceae
Fermentation products, Acetate + Hydrogen
Carbon dioxideNH4+
N2 NO3-
NO2-AzoarcusBurkholderiaAnammox
Syntrophy innitrate-reducingenrichmentcultures(SwampandCartwright)
BenzenePeptococcaceae
Fermentation products, Acetate + Hydrogen
Carbon dioxideNH4+
N2 NO3-
NO2-AzoarcusBurkholderiaAnammox
BenzoateAzoarcus
, Toluene or Phenol
CulturebecomesdominatedbyAzoarcus iffedbenzoate,tolueneorphenol
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CulturebecomesdominatedbyAzoarcus iffedbenzoate,tolueneorphenolwithnitrate
Anaerobic Pathways clearly established in Azoarcus
Rela
tive
abun
danc
e
AzoarcusAzoarcusAzoarcus
Pepto
The anaerobic benzene carboxylase gene neighborhood in Peptococaccea
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+ CO2AbcDA
COOH
+ CoA
COSCoA
Putative carboxylase
BzlA
Prenyl TransferaseBUT: Not yet biochemically proven!
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Figure 6: Enzyme activity of UbiX, a prenyltransferase. UbiX bindsoxidized Flavin mononucleotide (FMN) to Dimethylallyl phosphate(DMAP), producing prenylated FMN (prFMN), an important cofactorfor UbiD in the ubiquinonecycle (White et al., 2015)
Nature 2015
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Figure 6: Enzyme activity of UbiX, a prenyltransferase. UbiX bindsoxidized Flavin mononucleotide (FMN) to Dimethylallyl phosphate(DMAP), producing prenylated FMN (prFMN), an important cofactorfor UbiD in the ubiquinonecycle (White et al., 2015)
UbiX is a prenyltransferase:It adds a prenyl group to flavinmononucleotide (FMN)to form prenylated FMN – an important co-factor for ubiquinone biosynthesis
White et al. (2015) Nature, 522(7557): 502
Proposed!
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The Putative Anaerobic Benzene Carboxylase Neighborhood: Metagenomic, Metatranscriptomic and Metaproteomic Analysis
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Other proteins FeS binding proteins Transcriptional regulators UbiD-like proteins UbiX-like proteins
Expressed genes (proteomic analysis)
transcriptome
UbiD/UbiX Involved in Biodegradation of Aromatic Contaminants
Peptococcaceae_benEnrichment culture BF
ubiX(N
RBC0
004)
abcA
Geobacter Metallireducens
ubiX(Q
5P48
3)
ppsA
ppsB
ppcB pp
sC
ppcB
ppcCpp
cAppcD pp
sC ppsB pp
sA
Azoarcus sp. EbN1
ubiX(Q
39TU
0)
phthaloyl-CoA
decarboxylase
ubiX(D
5NWH
5)
CoA
trans
feras
e
CoA
trans
feras
e
Azoarcus sp. EbN1
1kb
Benzene
Phenol
Phthalate
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1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
Gen
e co
pies
/ml m
icro
cosm
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
Biostimulation with Nitrate: Emergence of Indigenous Benzene-Degrader
0.000
0.005
0.010
0.015
0 50 100 150 200
benzene
abcA
Total bacteria
21 187138
0.000
0.005
0.010
0.015
0 50 100 150 200
Benz
ene
(mm
ol/b
ottle
)
18713821
Day Day
Nitrate-amendment 1 Nitrate-amendment 2
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Delta_ORM2: below detection limit
Pepto-ben
2. Methanogenic CultureRefer to previous presentation by Fei Luo; Active cultures contain Deltaproteobacterium ORM2. Genomic and Proteomic data; No evidence for AbcDA, nor UbiX
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2008 - Dual Isotope Plot Suggested two Mechanisms
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Mancini, S. A., Devine, C. E., Elsner, M., Nandi, M. E., Ulrich, A. C., Edwards, E. A., & Sherwood Lollar, B. (2008). Isotopic Evidence Suggests Different Initial Reaction Mechanisms for Anaerobic Benzene Biodegradation. Environmental Science & Technology, 42(22), 8290–8296.
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Culture scale-up for potential use for bioaugmentation
http://anaerobicbenzene.ca/
Methanogenic Enrichment culture OR-CH4
Scaled up to 100L:DGG-1
Plan to test at a field site in 2018
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Dunja Grbic-Galic (1949-1993)
• Professor; Stanford University Environmental Engineering and Science
• Anaerobic microbiology• Ferrulate (lignin)• Toluene & Benzene from
petroleum hydrocarbons• Kindness
DGG: Dunja Grbic-Galic
Conclusions• Anaerobic benzene degradation appears limited to a few groups of
organisms that we can now track. Could these organisms also be specialists (like Dehalococcoides) offering hope for bioaugmentation?
• Peptococcaceae-containing benzene-degrading cultures appear specific to slightly higher redox like nitrate-reducing & iron-reducing conditions. Initial step likely a carboxylation supported by a modified (prenylated?) FMN cofactor, although biochemical evidence lacking
• Deltaproteobacteria (ORM2)-dominated cultures are inhibited by nitrate. These grow well under both under methanogenic and sulfate-reducing conditions. Mechanism still unknown
• The pathways for anaerobic benzene oxidation are still not confirmed, though genomic and proteomic tools are providing new leads 27
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Thank you!
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