Biodegradation and Detoxification

June 3, 2014

Joonhong Park

Definitions

Terms

a. Disappearances of a polluting chemical

b. Abiotic transformation of a chemical

c. Biotic transformation of a chemical (Biodegradation)

d. Detoxification

e. Activation

f. Cometabolic vs. Metabolic Biodegradation

Detoxification

Detoxification

The most important role of microorganisms in the transformation of pollutants

Detoxification refers to the change in a molecule that renders it less harmful to one ore more susceptible species (human, animals, plants and microorganisms)

Toxin

ToxinInactiveProduct

Metab.Seq. A

Metab.Seq. B

AtypicalProduct

CO2

TypicalProduct

DetoxificationReaction

Mineralization

Transformation processes resulting in detoxification

a. Hydrolysis: addition of water

b. Hydroxylation: addition of OH

c. Dehalogenation: removal of Cl or halogens; (i) reductive dehalogenation, (ii) hydrolytic dehalogenation, and (iii) dehydrodehalogenation

d. Demethylation or other dealkylation

e. Methylation: R-OH => R-O-CH3

f. Nitro reduction: R-NO3 => R-NH2

g. Deamination: metamitron, Figure 4.5

h. Ether cleavage: 2,4-D, Figure 4.5

i. Conversion of nitrile to amide: R-C≡N => R-C(=O)-NH2

j. Conjugation: A product of a toxicant combined with a natural metabolite

Activation

Activation

One of the more surprising, and possibly the most undesirable, aspects of microbial transformations in nature is the formation of toxicants.

Often, by-products of biodegradation are more toxic than their parental compounds

Important to know about the pathway of biodegradation including activation and its degradation products (intermediates)

Mechanisms of Activation (I)

a. Dehalogenation:PCE => TCE => DCE => VC => ethaneb. Halogenation: fungi, algae and bacteria add chloride or

bromine to organic compoundsc. Nitrosamine formation

R R NH + NO2

- = N-N=O + OH- R’ R’ (nitrosamine)

d. Epoxidation O -HC=CH- => -HC-CH- (ex. Aldrin to Dieldrin)

e. Conversion of phosphorothinate to phosphateRO S RO ORO -P-OX => RO - P – OX (cf. phosphorodithoates)

Mechanisms of Activation (II)

f. Metabolism of Phenoxyalkanoic Acids: beta-oxidation (2,4-D, Figure 5.5; 4-nonlyphenol Figure 5.6)

g. Oxidation of Thioethers O O -C-S-C- => -C-S-C => -C-S-C- Oh. Hydrolysis of Esters O O RCOR’ + H2O => RCOH + HOR’

i. Other Activations - Production of TCDD by peroxidases - Methylation (organics, mercury, arsenic, tin etc.) - Demethylation - Anaerobic degradation of RDX into 1,1-and1,2-dimethylhydrazine

Discussion: Risks of Biodegradation

The products of microbial biodegradation of a pollutant may or may not be more toxic to human, animals, plants or other ecosystems.

Is biodegradation always good?

What should be the ultimate goal of bioremediation?

What measurements are required for answering the above questions?

Effect of Chemical Structure on Biodegradation

Chemical Factors Affecting Biodegradation

Many possible chemical reactions are foreign to microorganisms (xenobiotics)

Biodegradation rates vs. Xenobiotic metabolism(growth-linked metabolism vs. cometabolism)

Chemical Factors Affecting Biodegradation

Xenophores (substituents): Table 11.2 in B&B- Cl, Br, NO2, SO3H, CN or CF3 (strong xenophores)- CH3, NH2, OH and OCH3 (often acting as xenophores)- OH, COOH, amide, anhydride etc. (stimulaitng

biodegradation)

The addition of xenophore: Table 11.2 in B&B- The more addition, the less biodegradable.

The position of xenophore: Table 11.3 in B&B

Chemical Factors Affecting Biodegradation

Methylbranching vs. beta-oxidation-Linear ABS vs. ABS with a quaternary C

Molecules containing an aromatic and alky/aliphatic acid moiety

PAH biodegradability (Figure 11.5 in B&B): 3 rings (biodegradable) vs. 4 and more rings (little biodegradable)

Chlorinated Hydrocarbon Biodegradability

Degree of Halogenation vs. Biodegradation

C C

Cl

Cl

Cl

Cl

C C

Cl

Cl

H

Cl C C

H

Cl

Cl

H

C C

Cl

Cl

H

H

C C

H

H

Cl

H

C C

H

H

H

H

Tetrachloroethene(Perchloroethene, PCE)

Trichloroethene(TCE, Cs = 1,100 mg/L)

cis-Dichloroethene(cDCE)

trans-Dichloroethene(tDCE)

monochloroethene(vinyl chloride, 발암물질 )

ethene

가장 산화됨

가장 환원됨

Monochlorinated Polychlorinated0.25 4

Degree of Chlorination

So

rpti

on

on

to S

ub

surf

ace

Mat

eria

l

Deg

rad

atio

n R

ateSorption

Reductive dechlorination

Aerobic degradation

BiodegradabilityThe apliphatic and aromatic hydrocarons are readily biodegradable by a

range of aerobic bacteria and fungi. The key is that molecular O2 is needed to activate the molecules via initial oxygenation reactions.

Evidence of anaerobic biodegradation of aromatic hydrocarbons is growing. Anaerobic biodegradation rates are slower than aerobic rates, but they can be important when fast kinetics are not essential.

Most halogenated aliphatics can be reductively dehalogenated, although the rate appears to slow as the halogen substituens are removed.

Highly chlorinate aromatics, including PCBs, can be reductively dehalogenated to less halogenated species.

Lightly halogenated aromatics can be aerobically biodegraded via initial oxygenation reactions.

Many of the common organic contaminants show inhibitory effects on microorganism growth and metabolism. Due to their strongly hydrophobic nature, many of the inhibitory responses are caused by intereactions with the cell membrane. In some cases, intermediate products of metabolism can be more toxic than the original contaminant.

Predicting Biodegrability?Structure-Activity Relationship (SAR) or

Structure-Biodegradation Relationship (SBR)

Biodegradation rate of a compound = f(waster solubility, melting/boiling points, molecular weight, molecular volume, molar refractivity, density, logKow, van der Waals radius, van der Waals volume, Hammett’s sigma, pKa, dipole moment, etc.)

Microbial Physiology

Microbial Ecology

Environmental Abiotic Factors

Acclimation

Factors Affecting Acclimation

Environmental Factors (temperature, pH and aeration)

The concentration of N, P or both (nutrients) (cf. the degradation of P- and N-containing organic

compounds is inhibited in the presence of nutrients)

The concentration of the compound that is being metabolized- Threshold- Detection limit

Site specific microbial communities- Biodegradation population- Interaction with other population members

Explanations for the Acclimation Phase

(a) Proliferation of small populations (Figure 3.3 in B&B)

(b) Presence of toxins (Figure 3.4 in B&B)

(c) Predation by protozoa (Figures 3.5 and 3.6 in B&B)

(d) Appearance of new genotypes (mutation or gene transfer)

(e) Enzyme Induction and Lag Phase (the regulations of gene expression and enzyme induction, diauxie, catabolic repression, etc.)

Predicting Biodegradation Intermediates (Pathways)

Biodegradation Pathway Databases

Biocatalysis/Biodegradation Database (Univ. of Minnesota)

http://umbbd.ethz.ch/index.html

List of 219 pathways, 1503 reactions, 1396 compounds993 enzymes, 543 microorganism entries, 249 biotransformation rules

KEGG Pathway Database – GenomeNet

http://www.genome.jp/kegg/pathway.html

Mono-cyclic Aromatics (BTEX): Upstream

Johnson, Park, Kukor and Abriola, 2006, Biodegradation

Hydroxylation via mono- and di-oxygenases

Ketone formation

Ring cleavage

Mono-cyclic Aromatics (BTEX): Upstream

Carboxylation (anaerobic conditions)

Mono-cyclic Aromatics (BTEX): Downstream

Mono-cyclic Aromatics (BTEX):

Reduction of Double Bonds

Benzene Cyclohexene

COOH COOH COOH

Benzoic acid 1-cyclohexenecarboxylic acid

cyclohexanecarboxylic acid

CH3

Toluene 4-methycyclohexanol

CH3HO

Cycloalkanes:

oxidation

-CH2-CH2- -CH-CH2-

OH

-C-CH2-

O

-HC=CH-

Hydroxylation

Ketone formation

Dehydrogenation

Methyl Groups (RCH3):

(terminal) oxidation

R-CH3 R-CH2

OH

R-CH

O

Alcohol

Aldehyde

Carboxylic acid R-C-OH

O

R could be alkanes and aromatics in oil products, surfactants etc.

Alkanes (CH3(CH2)nCH3):

Dehydrogenation

R-CH2-CH2-R’ R-CH=CH-R’

Ex. The conversion of eicosane (C20H42) to eicos-9-ene in soil The formation of 1-dodecene from tetradecane by anaerobes

Alkyl Groups (R(CH2)nCH3):

Sub-terminal Oxidation (hydroxylation or keto formation)

Ar-CH2-CH3

Ar-CH2-Ar

Ar-CH-CH3

O

Ar-CH-Ar

ethylbenzene

O

Alkenes and Others with Double Bonds:

Reduction, Oxidation, Hydration and Epoxide Formation

R-CH2-CH2-R’ R-CH=CH-R’

R-COH-CH2-OH R-CH=CH2

R’ R’

R-CHCH2-OH R-CH=CH2

R’ R’

R-CH-CH-R’ R-CH=CH-R’

O

Alkynes and Others with Triple Bonds:

Reduction

R-CH=CH2R-C≡CH

HC≡CH H2C=CH2

acetylene ethene (ethylene)

The acetylene reduction is done by bacterial nitrogen fixation

Carboxylic Acids (R-COOH):

Decarboxylation and Reduction

ArCOOH

CH3 Decarboxylation

ArH

R-CH-COOH R-CH2CH3

R-COOH R-CHO R-CH2OH

Reduction

Carboxylic Acids and Alcohols:

Ester Formation

R’-COOHR-CH2OH +

Ester

R-CH2OCR’

O

Esters: Hydrolysis

R’-COOHR-CH2OH +R-CH2OCR’

O

Microbial hydrolysis of malathion, bromoxynil, etc.

Alkanoic Acids [R(CH2)nCOOH], Alkanes [H(CH2)nCH3], and Alkyl Groups [R(CH2)nCH3]:

Beta-oxidation

CH3-COOH

R-(CH2)nCH2CH2COOH R-(CH2)nCH=CHCOOH

R-(CH2)nCHCH2COOH

OH

R-(CH2)nCCH2COOH

O

β-position

R-(CH2)nCOOH

+

Hydroxyl Groups (ROH):

Methylation and Ether (ROR’) Formation

R-OH R-OCH3

ArOCH2COOH

Ar ArOH ArOCH3 PAH

ArOH ArOCH3Phenoxyherbicides

ArOH ArOCH2CH3 O-ethylation

CH3

Ar

Ar

CH-O-CH

Ar

Ar

Ar

Ar

diphenylmetahne to1,1,1’1’-tetraphenyl-dimethyl ether

Ether (ROR):Cleavage

Table 12.9

Halogenated Aromatics:

Reductive Dehalogenation

Cl Cl

Cl

Cl

Cl

Cl

Cl Cl

Cl

Cl

Cl

H

+ [2H]

+ H+ + Cl-

Cl Cl

Cl

Cl

H

H

Cl Cl

H

Cl

H

Cl

Cl H

Cl

Cl

H

Cl

isomers

ArCl6 => HArCl5 => H2ArCl4 => H3ArCl3 => H4ArCl2 => H5ArCl

Halogenated Alkanes and Alkenes:

Reductive Dehalogenation

CCl4 => HCCl3 => H2CCl2

CCl4 + [2H] HCCl3 + H+ + Cl-

Cl2CHCHCl2 => Cl2CHCH2Cl => ClCH2CH2Cl => ClCH2CH3

C C

Cl

Cl

Cl

Cl

C C

Cl

Cl

H

ClC C

H

Cl

Cl

H

C C

Cl

Cl

H

HC C

H

H

Cl

H

C C

H

H

H

H

PCE

TCE

cDCE

tDCE

MCE (VC)

ethene

Halogenated Compounds:Hydrolytic Dehalogenation, Dehydrodehlogenation, Halogen Migration

R2CHCCl3 R2C=CCl2

RCl + H2O ROH + H+ + Cl- Hydrolytic Dehalogenation

Ex. Trans-1,3-dichloropropen into trans-3-chloroallyl alcohol

+ H+ + Cl-

R2CHCHCl2 R2C=CHCl

R2CHCH2Cl R2C=CH2

+ H+ + Cl-

+ H+ + Cl-

Dehydro-Dehalogenation

Ex. DDT metabolism (DDT=>DDE=>DDMU)

C C

Cl

Cl

H

ClTCE

C

Cl

Cl

H

Cl 1,1,1-trichloro-ethanol

C OH

H

HalogenMigration

Other Halogenated Compounds

ArCl ArSCH3

From THM (trihalomethyl)-containing compounds

RCCl3 RCOOH

To methylthio derivatives

Amines

Reductive deamination

RCH2CHCOOH RCH2CH2COOH

NH2

RCH2CHCOOH

OHHydrolyticdeamination

RCH=CHCOOH

Dehydro-deamination

N-methylation

N-acylation

ArNH2 ArNHCCH3

O

N-oxidation

RNH2 RNHOH RNO RNO2

N heterocycle

Aniline

2-methylquinoline

Amines

R-NH + NO2- R-N-NO + OH-

Dimerization

ArNH2 ArN=NAr

S-addition

RN-Alk2 RNH2

R’ R’

N-nitrosation

ArNH2 ArNHSCH3

O

Dealkylation

RN-Alk

ArN=NAr’ ArNH2 + Ar’NH2

Reduction

Carbamates and Amides: Cleavage

Carbamate Cleavage

RCNHR’ RCOOH + H2NR’

O

Amide Cleavage

RCNH2 RCOOH + H3N

O

Nitriles (RC≡N)

RC≡N

Dihalogenated benzonitriles used as pesticides

RCNH2

O

RCOH

O

Azobenzenes: Reduction to Amines

ArN=NAr’ ArNH2 + H2NAr’

N-Nitroso Compounds (Nitrosamines):

R-N-NO R-N-H

R’ R’Denitrosation

Nitro Compounds (RNO2)

ArNO2

Reduction

Hydrolytic Denitration

ArNO ArNHOH ArNH2(nitroso-) (hydroxylamino-) (amino-)

RNO2 + H2O ROH + NO2- + H+

ArNO2

Reductive Denitration

ArH

Nitrate Esters (RONO2)Cleavage RONO2 ROH

R(ONO2)3 HOR(ONO2)2 (HO)2RONO2 (OH)3R

C-S Bond: Cleavage

Thiols (RSH):

ArSH ArSCH3 RSH RSSR

Sulfate Esters (ROSO3H):Cleavage

ROSO3H ROH

Thioethers (RSR’): Oxidation

RSR’ RSR’ RSR’ O O

O

= ==

Disulfides(RSSR): Cleavage

RSSR RSH

RSR’ RSH (+ HR’) RSR’ ROH

Methylation Dimerization

CHSR’’ C=O RSO3H ROHR

R’

R

R’

Phosphate Esters

PhoshorothioatesCleavage

P-ORAlkO O

AlkO

=

P-ORAlkO

HO

=O

HOP-OR

HO O=

POHAlkO O

AlkO=

P-ORAlkO S

AlkO

= P-OHAlkO S

HO

=

P-OHAlkO S

AlkO

=

P-OHAlkO O

HO=

P-OHAlkO O

AlkO

=

PSRAlkO O

AlkO

=

Degradation

PSHAlkO O

AlkO

=

P-OHAlkO O

AlkO

=

Phoshoro-Di-thioatesCleavage

P-SRAlkO S

AlkO

=

PSRAlkO S

HO

= P-SHAlkO S

AlkO

=

P-OHAlkO O

AlkO

=

PhoshonatesCleavage

RP-OH + H2O RH + HOP-OHS

OH

=

OH

= O

Triarly PhoshonatesCleavage

ArOP-OAr ArOPOAr + ArOHO

OAr

=

OH

= O

P=SConversion to P=O

PSRAlkO S

AlkO=

PSRAlkO O

HO

=

Addition Reactions

ArH ArCH3

ArH ArNO2

CHCCl3

ROH + H2SO4 ROSO3H + H2O

Ar

ArCHC≡N

Ar

Ar

ArNO2

ArSCH3

Methyl-

Nitro-

Nitrile

Sulfonate

Methylthio-