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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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