Biodegradation

Dr. Stephen Johnson

s.j.johnson@gre.ac.uk

Fate of an organic contaminant

Volatilization Leaching Sequestration Bioaccumulation Biodegradation

Bio – X - ation

Biodeterioration– BAD

Biodegradation– NEUTRAL

Bioremediation– GOOD

Microbial biodegradation

Aerobic Anaerobic In situ Ex situ Microbes + contaminants + TEA -> CO2 +

H2O + biomass

Requirements for life

Energy Water Carbon Nitrogen Oxygen? Phosphate Trace elements http://www.abe.iastate.edu/Ae573_ast475/

Stoichiometry_Notes.htm

Redox

In most cases the contaminant is oxidised (loses electrons). For this to happen, another compound needs to be reduced (gain electrons) to prevent electrons from accumulating. Usually there is a chain of these redox couples with the electrons eventually being taken up by a terminal electron acceptor

Oxygen CO2 Mn(IV) Mn(III) NO3

- NO2-

Fe(III) Fe(II) SO4

2- H2S H CH4

Hydrocarbon degradation

Aerobic Nitrate Manganese Fe(III) Fe(II) Sulphate Methanogenesis

Benzene

Toluene Ethylbenzene

m-Xylene p-Xylene o-Xylene Alkanes Alkenes LAB PAH Others

Redox zones

Vadose Zone

Saturated Zone

Bedrock

Water table

Aerobic

MethanogenicSulphate

Nitrate

Aerobic degradation ofn-alkanes

-oxidation Degrades hydrocarbon (fatty

acid) chain Removes 2 carbons at a time Ubiquitous pathway BUT needs O2

SH CoA

CH3

C97

S

O

CoA

OH2

SH CoA

CH2

112

CH

2

CH2

C115

O

O

R

RCH2

CH

2

CH2

C12

S

O

CoA

RCH2

CH

CH

S

O

CoA

RCH2

CH

CH2

C42

S

O

CoA

OH

RCH2

C56

CH2

C58

S

O

CoA

O

RCH2

C92

S

O

CoA

Oxidation

Hydration

Oxidation

Thiolysis

+

Activation

Fatty acid

Acyl CoA

Enoyl CoA

L-Hydroxyacyl CoA

Ketoacyl CoA

Acyl CoA Acetyl CoA

Beta oxidation (Adapted from Stryer 1981)

Initial anaerobic transformations of toluene

CH3

Toluene

CH3

OH

o-Cresol

CH3

OHp-Cresol

CH2OH

Benzyl Alcohol

CH3

Methylcyclohexane

Ring reduction/Ring cleavage/Mineralizationof Aliphatics

Anaerobic mineralizationof toluene

CH3

Toluene

CH2

CH

2

Hydrocinnamoyl-CoA

CH2

SCoA

O

O

Benzoyl-CoA

S

CoA

CoASH

CH2

CH C

H2

SCoA

O

B-hydroxycinnamoyl-CoA

OH

CH2

CH

CH

SCoA

O

Cinnamoyl-CoA

SCoA

O

CH2 CH2

SCoA

OO

B-ketocinnamoyl-CoA

CO2

OH2

SCoA

O

Proposed pathway for anaerobic toluene mineralization - after Chee-Sanford et al 1996

2e-, 2H+ 2e-, 2H+

2e-, 2H+

Anaerobic degradation of toluene

CH3

Toluene

CH2

CH

Benzyl-succinate

CH2

OO

O

O

O

Benzoyl-CoA

S

CoA

CoASH Succinyl-CoA

CH CH2

O

O

O

E-Phenylitaconyl-CoA

S

CoA

CH C

H CH2

O

O

O

2-Carboxymethyl-3-Hydroxy-Phenylpropionyl-CoA

OH

S

CoA

OH2

CH C

H2

O

O

O

O

S

CoA

Benzylsuccinyl-CoA

CH2

CH C

H2

O

O

OS

CoA

Benzylsuccinyl CoA

BenzylsuccinateSynthetase

Benzylsuccinate-CoA Transferase Benzylsuccinyl-CoA

Dehydrogenase

3-Hydroxyacyl-CoADehydrogenase

Benzoylacetyl-CoAThiolaseolase

Proposed pathway for anaerobic toluene degradation - after Heider et al 1999

FumarateSuccinyl CoA Succinate

Phenylitaconyl-CoAHydratase

2[H]

2[H]

Anaerobic ethylbenzene degradation

CH2

CH3

Ethylbenzene

CH CH3OH

1-Phenylethanol

CH3O

Acetophenone

O O

CH2O

Benzoylacetate

O O

CH2O

SCoA

Benzoylacetate-CoA

O S

CoA

Benzoyl-CoA

OH2 CO2 CoASH CoASH Acetyl-CoA

EthylbenzeneDehydrogenase

1-PhenylethanolDehydrogenase

AcetophenoneCarboxylase

Benzoylacetyl-CoAforming enzyme

Benzoylacetyl-CoACoA thiolase

Proposed pathway for anaerobic ethylbenzene degradation - after Heider et al 1999

2[H]2[H]

Anaerobic alkylbenzene degradation

Toluenem-Xylene

p-Xylene

Ethylbenzeneo-Xylene

COOH

COOH

COOH

COOH

CH3

COOH

COOH

CH3

COOH

COOH

CH3

COOH

COOH

CH3

CH3

COOH COOH

CH3

COOH

CH3

CH3

COOHCOOH

CH3

COOH

CH3

COOH

COOH

COOH

COOH

Elshahed et al 2001

Alkylbenzenes (T,E,X) Key metabolites in

degradation All seen in

laboratory/ground water

Benzoate

Anaerobic benzoate degradation

SCoAO SCoAO

SCoAO SCoAO

OH

SCoAO

OCOO-

SCoAO

COO-

SCoAO

COO-

SCoAO

OH

SCoAO

OH

SCoAO

OH OH

SCoAO

OH O

OHO

B e n z o y l - C o A

P i m e l y l - C o A

C y c l o h e x - 1 , 5 - d i e n e1 - c a r b o x y l - C o A

6 - H y d r o x y c y c l o h e x - 2 - e n e -1 - c a r b o x y l - C o A

C y c l o h e x - 1 - e n e1 - c a r b o x y l - C o A

2 - H y d r o x y c y c l o h e x a n e -1 - c a r b o x y l - C o A

2 - K e t o C y c l o h e x a n e -1 - c a r b o x y l - C o A

2 , 3 - D e d e h y d r o -p i m e l y l - C o A

3 - H y d r o x y p i m e l y l - C o A

2 , 6 - D i h y d r o x y c y c l o h e x a n e -1 - c a r b o x y l - C o A

6 - O x o - 2 - h y d r o x y c y c l o h e x a n e -1 - c a r b o x y l - C o A

E1

E2E3 E4 E5 E6

E7

E8

E9 E10

E11

E1 -- Benzoyl-CoA reductase

E2 -- Cyclohex-1,5-diene -carboxyl-CoA reductase

E3 -- Cyclohex-1-ene 1-carboxyl-CoA hydratase

E4 -- 2-Hydroxycyclohexane-1-carboxyl-CoA dehydrogenase

E5 -- 2-Ketocyclohexane11-carboxyl-CoA hydrolase

E6 -- Pimelyl-CoA dehydrogenase

E7 -- 3-hydroxyacyl-CoA deyhdratase

E8 -- Cyclohex-1,5-diene-1-carboxyl-CoA hydratase

E9 -- 6-Hydroxycyclohex-2-ene-1-carboxyl-CoA hydratase

E10 -- 2,6-Dihydroxycyclohexane-1-carboxyl-CoA dehydrogenase

E11 -- 6-Oxo-2-hydroxycyclohexane-1-carboxyl-CoA hydrolase

Harwood and Gibson (1997) and Koch et al. (1993)

B e n z o a t e

Anaerobic degradation of n-alkanes

Limited range of chain lengths No < 6 C to date Pathways unknown Specific to organism May involve addition/removal of

odd number of C Rate of dissolution may limit rate

of degradation

Aerobic degradation of LAB

If chain > 3 long then starts with -oxidation of methyl terminus/i

Ring cleavage by oxidationR

CH

CH

R

OH

OH

R

OH

OH

R

OH

COOHO

O

COOH

RCOOH

NAD+ NADHNADH NAD+

O2 O2

+

Alkylbenzene Dihydrodiol Ring fissionproduct 2-Oxopenta-

4-enoate

2,3-Dihydroxy-alkylbenzene

Smith & Ratledge 1989

E2 E3 E4E1

E1 = Alkylbenzene dioxygenase

E2 = cis-alkylbenzene glycol dehydrogenase

E3 = 2,3-dihydroxyalkylbenzene 1,2-dioxygenase

E4 = ring fission product-hydrolysing enzyme

Anaerobic degradation of LAB

-oxidation? Conversion to benzoyl CoA? Hydrolytic ring cleavage? Limited by rate of dissolution?

Generalized breakdown

HYDROCARBON (eg BTEX)

Aerobic

Chain degraded by Beta oxidation

Convert to e.g.benzoyl CoA

Ring cleavage by hydrolysis (add H2O)

Anaerobic

?

Ring cleavage by oxygenases (add O2)

Organisms

Bacteria– bioremediation

Fungi– mycoremediation

Plants– phytoremediation

Bioavailability

May not be available to organisms Chemically Physically

– Solubility– Sorption

Pathways

The University of Minnesota Biocatalysis/Biodegradation Database– http://umbbd.ahc.umn.edu/

Bioremediation techniques

MNA/MENA Landfarming Bioventing/sparging Windrows Composting Biopile Biofiltration Bioaugmentation Biostimulation Redox/TEA

Group 1Not degraded

Bitumen Asphalt Metals Inorganic acids Asbestos Complex cyanides

Group 2May be degraded in lab

Higher MW PAHs PCBs Tars

Group 3 Demonstrated but not regularly achieved

Explosives Pesticides (e.g. lindane, malathion, diuron,

mecoprop, paraquat) PCP High MW PAH Branching aliphatics (e.g. hopane) Surfactants (e.g.LAS) MTBE Complex cyanides (?)

Group 4Regularly treated aerobically

Diesel Jet fuel BTEX Paraffin Ammonia Crude oil Lubricating oil Petrol Phenol

Chlorophenols Organic acids Creosote Alcohols Aldehydes Ketones Some surfactants Some pesticides Low MW PAHs

Group 5Regularly treated anaerobically

Chlorinated solvents

Soil factors affecting degradation

Organic matter content Microbial activity pH

– ionisable compounds– Acid/base catalysed degradation

Temperature Soil water content Depth

– Faster near surface

Phytoremediation

Rhizoremediation Transpiration (control flow) Volatilisation Plant metabolism Accumulation Stabilisation/immobilisation