5. Requirements for life
Energy
Water
Carbon
Nitrogen
Oxygen?
Phosphate
Trace elements
http://www.abe.iastate.edu/Ae573_ast475/Stoichiom
etry_Notes.htm
6. 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)
SO42- → H2S
H → CH4
9. Redox zones
Vadose Zone
Methanogenic Water table
Sulphate
Nitrate
Aerobic Saturated Zone
Bedrock
10. Aerobic degradation of
n-alkanes
β-oxidation
Degrades hydrocarbon (fatty
acid) chain
Removes 2 carbons at a time
Ubiquitous pathway
BUT needs O2
11. O
H2
R C C
115
C C O
112 H H2
2
Fatty acid
HS CoA
Activation
O
H2
R C C
12
C C S
H2 H2
Acyl CoA CoA
Oxidation
O
H
R C
C C S
H2 H
Enoyl CoA CoA
H2O
Hydration
OH O
R C C
H 42
C C S
H2 H2
L-Hydroxyacyl CoA CoA
Oxidation
O O
R C C
56 58
C C S
H2 H2
Ketoacyl CoA CoA
HS CoA
Thiolysis
O O
R
C
C
92
S
+ H3C
C
97
S
H2
CoA CoA
Acyl CoA Acetyl CoA
Beta oxidation (Adapted from Stryer 1981)
12. Initial anaerobic
transformations of toluene
CH3
OH
o-Cresol
CH3 CH3
CH3
Ring reduction/
Ring cleavage/
Mineralization
of Aliphatics
Methylcyclohexane Toluene OH
p-Cresol
CH2OH
Benzyl Alcohol
13. Anaerobic mineralization
of toluene
O O
OH O
H2
H
C C
CH3 C
H2C C SCoA H2C C SCoA H
H2 H H2C C SCoA
H2
Toluene Hydrocinnamoyl-CoA Cinnamoyl-CoA
B-hydroxycinnamoyl-CoA
O
H2O
2e-, 2H+ 2e-, 2H+
SCoA
O O CoA
O S
H2C C SCoA
H2
CO2
B-ketocinnamoyl-CoA Benzoyl-CoA
2e-, 2H+
CoASH O
SCoA
Proposed pathway for anaerobic toluene mineralization - after Chee-Sanford et al 1996
14. Anaerobic degradation of
toluene
CoA CoA
O O O S O S
O O O
C C
CH3 H H
H2C C O H2C C O HC C O
H2 H2 H2
Benzylsuccinate Benzylsuccinate-
CoA Transferase Benzylsuccinyl-CoA
Synthetase Dehydrogenase
Toluene Benzyl-succinate Benzylsuccinyl CoA
E-Phenylitaconyl-CoA
Fumarate 2[H]
Succinyl CoA Succinate
CoA CoA
O S O S
O O CoA
HO C O C O S
H H
C C O H
C O
H2 H2
Phenylitaconyl-CoA 3-Hydroxyacyl-CoA
Hydratase Benzoylacetyl-CoA
Dehydrogenase Thiolaseolase
2-Carboxymethyl-3-Hydroxy-Phenylpropionyl-CoA Benzoyl-CoA
Benzylsuccinyl-CoA
H2O
2[H] CoASH Succinyl-CoA
Proposed pathway for anaerobic toluene degradation - after Heider et al 1999
15. Anaerobic ethylbenzene
degradation
CoA
S
O O O O
CoA
CH3 HO CH3 O CH3 O CH2 O CH2 O S
H
H2C C
Ethylbenzene 1-Phenylethanol Acetophenone Benzoylacetyl-CoA
Dehydrogenase Dehydrogenase Benzoylacetyl-CoA
Carboxylase forming enzyme CoA thiolase
Ethylbenzene Acetophenone Benzoylacetate Benzoylacetate-CoA Benzoyl-CoA
1-Phenylethanol
H2O 2[H] CO2
2[H] CoASH CoASH Acetyl-CoA
Proposed pathway for anaerobic ethylbenzene degradation - after Heider et al 1999
16. Anaerobic alkylbenzene
degradation
Alkylbenzenes (T,E,X)
Key metabolites in
Toluene o-Xylene Ethylbenzene
m-Xylene
p-Xylene
degradation
COOH COOH COOH COOH COOH
H3C
COOH COOH COOH COOH COOH
All seen in
CH3
CH3
laboratory/ground
CH3
COOH COOH COOH
water
CH3
CH3
CH3
Benzoate COOH
COOH
COOH
COOH COOH
COOH
CH3
CH3
CH3
COOH
Elshahed et al 2001
17. Anaerobic benzoate
degradation
O OH
O SCoA O SCoA O SCoA O SCoA O SCoA
B e n z o a t e
OH O
E3 E4 E5 COO- E6 COO-
E2
E7
O SCoA O SCoA O SCoA
C y c l o h e x - 1 - e n e y d r o x y c y c l o h e x a n e - o C y c l o h e x a n e P i m e l y l - C o A
2 - H 2 - K e t - 2 , 3 - D e d e h y d r o -
1 - c a r b o x y l - C o A - c a r b o x y l - C o A
1 1 - c a r b o x y l - C o A p i m e l y l - C o A
HO
E1 COO-
O SCoA O SCoA E11
E8 O SCoA
C y c l o h e x - 1 , 5 - d i e n e 3 - H y d r o x y p i m e l y l -
B e n z o y l - C o A 1 - c a r b o x y l - C o A
HO HO OH HO O
E9 E10
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 2 , 6 - D i h y d r o x y c y c l o h e x a n e - - O x o - 2 - h y d r o x y c y c l o h e x a n e -
6
1 - c a r b o x y l - C o A 1 - c a r b o x y l - C o A
E1 -- Benzoyl-CoA reductase E7 -- 3-hydroxyacyl-CoA deyhdratase
E2 -- Cyclohex-1,5-diene -carboxyl-CoA reductase E8 -- Cyclohex-1,5-diene-1-carboxyl-CoA hydratase
E3 -- Cyclohex-1-ene 1-carboxyl-CoA hydratase E9 -- 6-Hydroxycyclohex-2-ene-1-carboxyl-CoA hydratase
E4 -- 2-Hydroxycyclohexane-1-carboxyl-CoA dehydrogenase E10 -- 2,6-Dihydroxycyclohexane-1-carboxyl-CoA dehydrogenase
E5 -- 2-Ketocyclohexane11-carboxyl-CoA hydrolase E11 -- 6-Oxo-2-hydroxycyclohexane-1-carboxyl-CoA hydrolase
E6 -- Pimelyl-CoA dehydrogenase
Harwood and Gibson (1997) and Koch et al. (1993)
18. 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
19. Aerobic degradation of LAB
If chain > 3 long then starts with β-oxidation of methyl
terminus/i
Ring cleavage by oxidation
R R R R RCOOH
NADH NAD+ H OH
C NAD+ NADH OH O +
COOH COOH
E1 C
O2 H OH E2
OH O2
E3 OH E4
O
Alkylbenzene Dihydrodiol 2,3-Dihydroxy- Ring fission
alkylbenzene product 2-Oxopenta-
4-enoate
E1 = Alkylbenzene dioxygenase
E2 = cis-alkylbenzene glycol dehydrogenase
E3 = 2,3-dihydroxyalkylbenzene 1,2-dioxygenase
E4 = ring fission product-hydrolysing enzyme Smith & Ratledge 1989
20. Anaerobic degradation of
LAB
β-oxidation?
Conversion to benzoyl CoA?
Hydrolytic ring cleavage?
Limited by rate of dissolution?
21. Generalized breakdown
HYDROCARBON (eg BTEX)
Anaerobic
Aerobic
?
Chain degraded by Beta oxidation
Convert to e.g.benzoyl CoA
Ring cleavage by oxygenases (add O2) Ring cleavage by hydrolysis (add H2O)
Until about 1990, it was generally considered that hydrocarbons were essentially immune to anaerobic degradation. Since than, a large number of papers have been written about the degradation of, especially, BTEX compounds. Much of this has been centred around Derek Lovley at the University of Minnesota.
If a terminal isomer of LAB were degraded via this pathway, the end result would be either toluene or ethylbenzene, depending on whether the number of carbon atoms in the original alkane chain was odd or even. However, since the pathway requires the addition of molecular oxygen (at two points in each cycle) it will not occur in anaerobic conditions. In any event, the terminal isomers are absent in synthetic LAB. -oxidation of the isomers found in cable oil may lead to one of a number of structures. This will be dependant on whether the initial chain(s) are odd or even in length, whether both ends of the chain are attacked, and on how close to the phenyl group the relevant enzyme can operate before the charge or physical size of the group interferes too much. However, -oxidation does require molecular oxygen and so is unlikely to occur in strictly anaerobic conditions.
If a terminal isomer of LAB were degraded via this pathway, the end result would be either toluene or ethylbenzene, depending on whether the number of carbon atoms in the original alkane chain was odd or even. However, since the pathway requires the addition of molecular oxygen (at two points in each cycle) it will not occur in anaerobic conditions. In any event, the terminal isomers are absent in synthetic LAB. -oxidation of the isomers found in cable oil may lead to one of a number of structures. This will be dependant on whether the initial chain(s) are odd or even in length, whether both ends of the chain are attacked, and on how close to the phenyl group the relevant enzyme can operate before the charge or physical size of the group interferes too much. However, -oxidation does require molecular oxygen and so is unlikely to occur in strictly anaerobic conditions.
A number of authors have investigated the degradation of BTEX compounds Grbic-Galic 1991
Proposed by Chee-Sanford et al 1996 Note Benzoyl CoA
Heider et al 1991 Bemzoyl CoA
Heider et al 1999 Benzoyl CoA
Benzoate
Benzoate, or more specifically, benzoyl-CoA is a central metabolite in anaerobic degradation pathways of aromatic compounds and it’s degradation is fairly well understood. These pathways are proposed from studies of the phototropic bacterium Rhodopseudomonas palustris and the denitrifiers Thauera aromatica (Previously Pseudomona s sp strain K172) and Azoarcus evansii (Previously Pseudomonas sp strain K740) .
Aekersberg et al . were the first to show that hexadecane and other long chain alkanes could be degraded to CO 2 by a bacterial strain under sulphate-reducing conditions . There was some evidence that this strain produced membrane lipids with an odd number of carbon atoms when fed alkanes with an even number of carbon atoms. This suggests that the alkane chain undergoes the removal or addition of an odd number of carbon atoms in this organism, which contrasts with the strictly even removal of -oxidation. This even-to-odd transformation has not been seen in subsequently identified alkane-degrading anaerobes and each species is specific to a limited range of chain lengths, indicating that a range of novel pathways are used.
A generalised pathway has been elucidated for the aerobic catabolism of C2-C7 n -alkylbenzenes (i.e. terminal isomers) by Pseudomonas sp. . Alkylbenzenes with an n -alkyl chain of more than 3 carbons are initially attacked by - or – oxidation at the methyl terminus. It is possible that both methyl termini are attacked in non-terminal isomers.
-oxidation? No - needs oxygen Conversion to benzoyl CoA? Perhaps - depends on whether alkyl chain degraded Hydrolytic ring cleavage? Most likely (though possibly oxidation under nitrate-reducing conditions) Limited by solubility? May be a plus - low solubility may make for low mobility and low toxicity The most likely route for anaerobic degradation of LABs will probably be an initial attack on the alkyl chain(s) to form an acyl CoA, followed by a hydrolytic ring cleavage.