**Bioremediation of Chlorobenzoic Acids**

Blanka Vrchotová, Martina Macková, Tomáš Macek and Kateřina Demnerová

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56394

**1. Introduction**

Chlorobenzoic acids (CBAs) can be released into the environment from many different sources. One possible source of CBAs is usage as herbicides or insecticides in agriculture. As a herbicide was used 2,3,6-CBA [1]. CBAs may also be formed as intermediates during the degradation of same herbicides. Namely 2,6-CBA is formed as an intermediate during microbial degradation of dichlobenil [2], 2,5-CBA in the chlorambene degradation [3] or 4-CBA is a final degradation product of the insecticide DDT [4].

Another large group of substances from whose metabolism in living organisms including mammals arise CBAs, are drugs such as indomethacin, bupropion or benzafibrate. Indome‐ thacin is an anti-inflammatory drug used to reduce fever, pain, stiffness and swelling by inhibition of the production of prostaglandins, compounds that cause these problems. From the decomposition of indomethacin arises 5-methoxy-2-methylindoleacetic acid and the same quantity of 4-CBA [5]. In the case of antidepressant bupropion just small amount (0.3%) of 3- CBA is formed next to threohydrobupropion, erythrohydrobupropion and hydroxybupropion [6, 7]. Decomposition of bezafibrate, an anti-obesity drug, leads to the formation of equal quantity of 4-CBA and 4-(2-aminoethyl)-α,α dimethyl-benzeneacetic acid. [8].

Well known is the formation of CBAs during degradation of polychlorinated biphenyls (PCBs). PCBs are degraded by bacteria by the so called upper degradation pathway when CBAs are formed as final degradation products (Figure 1.) [10-12]. CBAs are also formed during degradation of PCBs by white rot fungi [13] [14]. The result of these metabolic pathways is a mixture of CBAs with different position and number of chlorine atoms on benzene ring in dependence of chlorination of the degraded PCB congeners [9].

The accumulation of this way formed CBAs in waste water or in soil can lead to the deceleration or inhibition of degradation of substances of which the CBAs are degradation products [15,

© 2013 Vrchotová et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 4-(2-aminoethyl)-α,α dimethyl-benzeneacetic acid. [8].

Czech Republic

vrchotob@vscht.cz

**1. Introduction** 

**Bioremediation of Chlorobenzoic Acids** 

Blanka Vrchotová, Martina Macková, Tomáš Macek, Kateřina Demnerová

Institute of Chemical Technology, Prague; Department of Biochemistry and Microbiology; Prague

Chlorobenzoic acids (CBAs) can be released into the environment from many different sources. One possible source of CBAs is usage as herbicides or insecticides in agriculture. As a herbicide was used 2,3,6-CBA [1]. CBAs may also be formed as intermediates during the degradation of same herbicides. Namely 2,6-CBA is formed as an intermediate during microbial degradation of dichlobenil [2], 2,5-CBA in the chlorambene degradation [3] or 4-CBA is a final degradation product of the insecticide DDT [4].

Another large group of substances from whose metabolism in living organisms including mammals arise CBAs, are drugs such as indomethacin, bupropion or benzafibrate. Indomethacin is an anti-inflammatory drug used to reduce fever, pain, stiffness and swelling by inhibition of the production of prostaglandins, compounds that cause these problems. From the decomposition of

hydroxybupropion [6, 7]. Decomposition of bezafibrate, an anti-obesity drug, leads to the formation of equal quantity of 4-CBA and

The next step after reductive dehalogenation is the degradation of benzoic acid. Benzoic acid can be anaerobically degraded by two different pathways [24]. The first one is initiated by reduction of benzoic acid aromatic ring. Subsequent ring cleavage and degradation of intermediates occurs by reactions similar to β-oxidation of aliphatic carboxylic acids [25]. In the second pathway is the molecule of benzoic acid activated by binding to the CoA. CoA remains bound to the molecule during all degradation steps. In this pathway is benzoate-CoA degraded to the acetyl-CoA [26]. Acetyl-CoA is then degraded in tricarboxylic acid cycle to

From previous it can be concluded that in anaerobic degradation is the molecule of CBA degraded to methane, CO2 and water with benzoic acid as intermediate (Figure 2.) [17].

Anaerobic degradation of CBAs can be done by pure cultures of microorganisms as well as by consortia of anaerobic microorganisms. The first pure strain that has been observed to be able to anaerobically degrade CBA was strain *Desulfomonile tiedjei* [27]. This strain is capable of 3- CBA dehalogenation, by this process reaching enough ATP for its growth. So it does not need additional source of energy. Another strain able to anaerobic degradation of CBA is strain *Desulfomicrobium escambiense* [22] and the aforementioned strain *Rhodopseudomonas palustris*

Aerobic microorganisms evolved many different degradation strategies for CBAs degradation. Microbial aerobic degradation of CBAs depends on bacterial strain as well as on the structure

CBAs can be degraded via chlorocatechole (*clc* degradation genes) [28, 29] or by hydrolytic dehalogenation with hydroxybenzoic acid as an intermediate like in 4-CBA degradation (*fcb* degradation genes) [30-32] or by 4,5-dioxygenation like in 3-CBA and 3,4-CBA degradation where is 5-chloroprotocatechuic acid formed (*cba* degradation genes) [33, 34] or by 1,2 dioxygenase reaction [9, 35] or by degradation involving the formation of gentisate as an

Strains able to aerobically degrade CBAs belong to the gram negative strains as *Rhodococcus* or *Bacillus* as well as gram positive strains like *Pseudomonas, Burkholderia* or *Achromobacter* [9].

HCl

COOH

CO2 +

CH4

OH

O

Bioremediation of Chlorobenzoic Acids http://dx.doi.org/10.5772/56394 3

3CH

H2

the CO2 and water.

DCP3 [23].

and chlorination of CBA.

COOH

Cln

HCl HCl HCl

**Figure 2.** Anaerobic degradation of chlorobenzoic acid adapted from [17].

**2.2. Aerobic degradation of chlorobenzoic acids**

intermediate of CBA degradation [36, 37].

Figure 1. Degradation pathway of polychlorinated biphenyls by aerobic organisms [9]. BphA – biphenyl-2,3-dioxygenase; BphB – biphenyldihydrodiol-dehydrogenase; BphC – 2,3-dihydroxy-biphenyl-1,2-dioxygenase; BphD – 2-hydroxychlor-6-oxo-6-phenylhexa-2,4-dienoathydrolase; I – polychlorinated biphenyl; II – dihydrodiol; III – 2,3-dihydroxychlor biphenyl; IV – 2-hydroxychlor-6-oxo-6-phenylhexa-2,4-dienoic acid; V – chlorobenzoic acid; VI – 2-hydroxychlor-2,4-dienoate Well known is the formation of CBAs during degradation of polychlorinated biphenyls (PCBs). PCBs are degraded by bacteria by **Figure 1.** Degradation pathway of polychlorinated biphenyls by aerobic organisms [9]. BphA – biphenyl-2,3-dioxyge‐ nase; BphB – biphenyl-dihydrodiol-dehydrogenase; BphC – 2,3-dihydroxy-biphenyl-1,2-dioxygenase; BphD – 2-hydrox‐ ychlor-6-oxo-6-phenylhexa-2,4-dienoathydrolase; I – polychlorinated biphenyl; II – dihydrodiol; III – 2,3-dihydroxychlor biphenyl; IV – 2-hydroxychlor-6-oxo-6-phenylhexa-2,4-dienoic acid; V – chlorobenzoic acid; VI – 2-hydroxychlor-2,4-di‐ enoate

16]. Therefore, it is important to understand bioremediation mechanisms of CBAs and to know the impact of other organisms, xenobiotics composition and the influence of other CBA isomers present in contaminated area that affect this process. formed during degradation of PCBs by white rot fungi [13] [14]. The result of these metabolic pathways is a mixture of CBAs with different position and number of chlorine atoms on benzene ring in dependence of chlorination of the degraded PCB congeners [9]. The accumulation of this way formed CBAs in waste water or in soil can lead to the deceleration or inhibition of degradation of

the so called upper degradation pathway when CBAs are formed as final degradation products (Figure 1.) [10-12]. CBAs are also

substances of which the CBAs are degradation products [15, 16]. Therefore, it is important to understand bioremediation mechanisms of CBAs and to know the impact of other organisms, xenobiotics composition and the influence of other CBA isomers

#### **2. Bacterial degradation of chlorobenzoic acids 2. Bacterial degradation of chlorobenzoic acids**

present in contaminated area that affect this process.

Bacterial degradation of CBAs can be done under aerobic or anaerobic conditions. Under anaerobic conditions is dechlorination the first step of degradation followed by degradation of the aromatic ring [17, 18]. Bacterial degradation of CBAs can be under aerobic or anaerobic conditions. Under anaerobic conditions is dechlorination the first step of degradation followed by degradation of the aromatic ring, the degradation of benzoic acid formed from CBAs by dechlorination [17, 18].

The strategy of CBAs degradation under aerobic conditions depends on the position of chlorine atom or atoms on the aromatic ring. Crucial step is the dechlorination. Dechlorination step can be before or after degradation of aromatic ring. This depends on the structure of CBA as well as on the enzymatic equipment of the bacteria.

#### **2.1. Anaerobic degradation of chlorobenzoic acids**

Initial step of microbial anaerobic degradation of CBAs is done by process named reductive dehalogenation. During the dehalogenation are from the more chlorinated CBA by dehydro‐ genation process formed less chlorinated CBAs or benzoic acid and chlorine is released as hydrochloric acid [19]. Time scale of this process depends on the number and position of the chlorine atoms in the molecule of CBA and on composition of anaerobic consortium or on strain with dehalogenation activity. Dehalogenation of CBAs can be performed under methanogenic [20], denitrification [21] or sulfate reducing conditions [22]. Also phototrophic bacteria capable of reductive dehalogenation of CBAs are known, e. g. *Rhodopseudomonas palustris* DCP3 [23], strain able to degrade under anaerobic conditions 2-CBA; 3-CBA; 4-CBA and 3,4-CBA. This strain is also capable of anaerobic degration of benzoic acid.

The next step after reductive dehalogenation is the degradation of benzoic acid. Benzoic acid can be anaerobically degraded by two different pathways [24]. The first one is initiated by reduction of benzoic acid aromatic ring. Subsequent ring cleavage and degradation of intermediates occurs by reactions similar to β-oxidation of aliphatic carboxylic acids [25].

In the second pathway is the molecule of benzoic acid activated by binding to the CoA. CoA remains bound to the molecule during all degradation steps. In this pathway is benzoate-CoA degraded to the acetyl-CoA [26]. Acetyl-CoA is then degraded in tricarboxylic acid cycle to the CO2 and water.

From previous it can be concluded that in anaerobic degradation is the molecule of CBA degraded to methane, CO2 and water with benzoic acid as intermediate (Figure 2.) [17].

**Figure 2.** Anaerobic degradation of chlorobenzoic acid adapted from [17].

16]. Therefore, it is important to understand bioremediation mechanisms of CBAs and to know the impact of other organisms, xenobiotics composition and the influence of other CBA isomers

**Figure 1.** Degradation pathway of polychlorinated biphenyls by aerobic organisms [9]. BphA – biphenyl-2,3-dioxyge‐ nase; BphB – biphenyl-dihydrodiol-dehydrogenase; BphC – 2,3-dihydroxy-biphenyl-1,2-dioxygenase; BphD – 2-hydrox‐ ychlor-6-oxo-6-phenylhexa-2,4-dienoathydrolase; I – polychlorinated biphenyl; II – dihydrodiol; III – 2,3-dihydroxychlor biphenyl; IV – 2-hydroxychlor-6-oxo-6-phenylhexa-2,4-dienoic acid; V – chlorobenzoic acid; VI – 2-hydroxychlor-2,4-di‐

Bacterial degradation of CBAs can be done under aerobic or anaerobic conditions. Under anaerobic conditions is dechlorination the first step of degradation followed by degradation

The strategy of CBAs degradation under aerobic conditions depends on the position of chlorine atom or atoms on the aromatic ring. Crucial step is the dechlorination. Dechlorination step can be before or after degradation of aromatic ring. This depends on the structure of CBA as well

Initial step of microbial anaerobic degradation of CBAs is done by process named reductive dehalogenation. During the dehalogenation are from the more chlorinated CBA by dehydro‐ genation process formed less chlorinated CBAs or benzoic acid and chlorine is released as hydrochloric acid [19]. Time scale of this process depends on the number and position of the chlorine atoms in the molecule of CBA and on composition of anaerobic consortium or on strain with dehalogenation activity. Dehalogenation of CBAs can be performed under methanogenic [20], denitrification [21] or sulfate reducing conditions [22]. Also phototrophic bacteria capable of reductive dehalogenation of CBAs are known, e. g. *Rhodopseudomonas palustris* DCP3 [23], strain able to degrade under anaerobic conditions 2-CBA; 3-CBA; 4-CBA

and 3,4-CBA. This strain is also capable of anaerobic degration of benzoic acid.

present in contaminated area that affect this process.

present in contaminated area that affect this process.

chlorobenzoic acid; VI – 2-hydroxychlor-2,4-dienoate

**Bioremediation of Chlorobenzoic Acids** 

4-(2-aminoethyl)-α,α dimethyl-benzeneacetic acid. [8].

2 Applied Bioremediation - Active and Passive Approaches

OH

Cl. Cl. Cl. Cl.

H H

OH

Cl .

O2 H2 O2 H2O

BphA BphB BphC BphD

II III IV

.

Czech Republic

vrchotob@vscht.cz

**1. Introduction** 

. Cl

I

enoate

Blanka Vrchotová, Martina Macková, Tomáš Macek, Kateřina Demnerová

Institute of Chemical Technology, Prague; Department of Biochemistry and Microbiology; Prague

Chlorobenzoic acids (CBAs) can be released into the environment from many different sources. One possible source of CBAs is usage as herbicides or insecticides in agriculture. As a herbicide was used 2,3,6-CBA [1]. CBAs may also be formed as intermediates during the degradation of same herbicides. Namely 2,6-CBA is formed as an intermediate during microbial degradation of dichlobenil [2], 2,5-CBA in the chlorambene degradation [3] or 4-CBA is a final degradation product of the insecticide DDT [4].

Another large group of substances from whose metabolism in living organisms including mammals arise CBAs, are drugs such as indomethacin, bupropion or benzafibrate. Indomethacin is an anti-inflammatory drug used to reduce fever, pain, stiffness and swelling by inhibition of the production of prostaglandins, compounds that cause these problems. From the decomposition of indomethacin arises 5-methoxy-2-methylindoleacetic acid and the same quantity of 4-CBA [5]. In the case of antidepressant bupropion just small amount (0.3%) of 3-CBA is formed next to threohydrobupropion, erythrohydrobupropion and hydroxybupropion [6, 7]. Decomposition of bezafibrate, an anti-obesity drug, leads to the formation of equal quantity of 4-CBA and

OH

Figure 1. Degradation pathway of polychlorinated biphenyls by aerobic organisms [9]. BphA – biphenyl-2,3-dioxygenase; BphB – biphenyldihydrodiol-dehydrogenase; BphC – 2,3-dihydroxy-biphenyl-1,2-dioxygenase; BphD – 2-hydroxychlor-6-oxo-6-phenylhexa-2,4-dienoathydrolase; I – polychlorinated biphenyl; II – dihydrodiol; III – 2,3-dihydroxychlor biphenyl; IV – 2-hydroxychlor-6-oxo-6-phenylhexa-2,4-dienoic acid; V –

Cl .

OH O

OH COOH

Cl .

Cl.

COOH

Cl

CH2 COOH

V

VI

OH

Well known is the formation of CBAs during degradation of polychlorinated biphenyls (PCBs). PCBs are degraded by bacteria by the so called upper degradation pathway when CBAs are formed as final degradation products (Figure 1.) [10-12]. CBAs are also formed during degradation of PCBs by white rot fungi [13] [14]. The result of these metabolic pathways is a mixture of CBAs with different position and number of chlorine atoms on benzene ring in dependence of chlorination of the degraded PCB congeners [9]. The accumulation of this way formed CBAs in waste water or in soil can lead to the deceleration or inhibition of degradation of substances of which the CBAs are degradation products [15, 16]. Therefore, it is important to understand bioremediation mechanisms of CBAs and to know the impact of other organisms, xenobiotics composition and the influence of other CBA isomers

Bacterial degradation of CBAs can be under aerobic or anaerobic conditions. Under anaerobic conditions is dechlorination the first step of degradation followed by degradation of the aromatic ring, the degradation of benzoic acid formed from CBAs by

as on the enzymatic equipment of the bacteria.

**2.1. Anaerobic degradation of chlorobenzoic acids**

of the aromatic ring [17, 18].

dechlorination [17, 18].

**2. Bacterial degradation of chlorobenzoic acids**

**2. Bacterial degradation of chlorobenzoic acids** 

Anaerobic degradation of CBAs can be done by pure cultures of microorganisms as well as by consortia of anaerobic microorganisms. The first pure strain that has been observed to be able to anaerobically degrade CBA was strain *Desulfomonile tiedjei* [27]. This strain is capable of 3- CBA dehalogenation, by this process reaching enough ATP for its growth. So it does not need additional source of energy. Another strain able to anaerobic degradation of CBA is strain *Desulfomicrobium escambiense* [22] and the aforementioned strain *Rhodopseudomonas palustris* DCP3 [23].

#### **2.2. Aerobic degradation of chlorobenzoic acids**

Aerobic microorganisms evolved many different degradation strategies for CBAs degradation. Microbial aerobic degradation of CBAs depends on bacterial strain as well as on the structure and chlorination of CBA.

CBAs can be degraded via chlorocatechole (*clc* degradation genes) [28, 29] or by hydrolytic dehalogenation with hydroxybenzoic acid as an intermediate like in 4-CBA degradation (*fcb* degradation genes) [30-32] or by 4,5-dioxygenation like in 3-CBA and 3,4-CBA degradation where is 5-chloroprotocatechuic acid formed (*cba* degradation genes) [33, 34] or by 1,2 dioxygenase reaction [9, 35] or by degradation involving the formation of gentisate as an intermediate of CBA degradation [36, 37].

Strains able to aerobically degrade CBAs belong to the gram negative strains as *Rhodococcus* or *Bacillus* as well as gram positive strains like *Pseudomonas, Burkholderia* or *Achromobacter* [9].

#### *2.2.1. Degradation of 2-chlorobenzoic acid*

In case of 2-CBA degradation there are known three different ways of degradation. All of them are about dioxygenation reaction catalyzed by 2-halobenzoate-1,2-dioxygenase (EC 1.14.12.13) (Figure 3.).

First 2-halobenzoate-1,2-dioxygenase is a two componential enzymatic system [39]. This 2 halobenzoate-1,2-dioxygenase has high homology with the toluate and benzoate-1,2-dioxy‐ genase. Two component 2-halobenzoate-1,2-dioxygenase has high affinity to 2-CBA but low

**Figure 4.** Degradation of 2,4-dichlorobenzoic and 2,5-dichlorobenzoic acid catalyzed by 2-halobenzoate-1,2-dioxyge‐

OH

OH

Cl

OH

5

OH

Bioremediation of Chlorobenzoic Acids http://dx.doi.org/10.5772/56394

4-chlorokatechol

Cl

Cl

OH

OH

Cl

HOOC

Cl

HOOC

Second 2-halobenzoate-1,2-dioxygenase is three componential [35] and this enzyme catalyzes

Next to 1,2-dioxygenase activity the enzyme 2-halobenzoate-1,2-dioxygenase has also 1,6 dioxygenase activity [9, 41]. Strain *Pseudomonas* sp. B-300 was in presence of glucose able to degrade 2-CBA to the 3-chlorocatechol [41].Whereas *Pseudomonas* sp. B-300 was cultivated with 2-CBA as the only carbon and energy source just catechol was identified. From that it is obvious that 1,6-dioxygenase reaction occurs only in much smaller degree than 1,2-dioxygenase

Dioxygenase reaction in the 1,6- position is followed by 3-chlorocatechol ring cleavage forming

Another metabolite identified in medium after cultivation of strain *Burkholderia cepacia* 2CBS with 2-CBA was 2,3-dihydroxybenzoic acid [42]. Strain *Burkholderia cepacia* 2CBS degraded most of 2-CBA by 1,2-dioxygenase reaction, 2,3-dihydroxybenzoic acid was in medium accumulated just in small quantities. Formation of 2,3-dihydroxybenzoic acid indicates that two-component 2-halobenzoate-1,2-dioxygenase of strain *Burkholderia cepacia* 2CBS has next to 1,2-dioxygenase activity also 2,3-dioxygenase activity (Figure 3.). 2,3-dihydroxybenzoic acid

to 4-CBA; 2,4-CBA and 2,5-CBA[9, 40].

COOH

2,5-dichlorobenzoic acid

Cl

2,4-dichlorobenzoic

acid

COOH

Cl

nase.

Cl

Cl

2-halobenzoate-1,2-dioxygenase

2-halobenzoate-1,2-dioxygenase

degradation of 2-CBA; 2,4-CBA and 2,5-CBA.

reaction or in the presence of a rich source of energy.

chloromuconic acid which is dehalogenated.

is the dead-end product [9].

**Figure 3.** Aerobic degradation of 2-chlorobenzoic acid by the enzyme 2-chlorobenzoate-1,2-dioxygenase [9, 38].

Main activity of 2-halobenzoate-1,2-dioxygenase is 1,2-dioxygenase reaction. In this reaction is 2-CBA degraded to catechol. During this reaction are released carbon dioxide and chlorine. Enzyme 2-halobenzoate-1,2-dioxygenase has also 1,6-dioxygenase activity. In 1,6-dioxygenase reaction is formed 3-chlorocatechol. Last activity of 2-halobenzoate-1,2-dioxygenase is 2,3 dioxygenase reaction leading to the formation of 2,3-dihydroxybenzoic acid. Latter two reactions proceed only in a small degree [9].

Degradation of 2-CBA by 2-halobenzoate-1,2-dioxygenase was reported in two groups of microorganisms. First group degraded only 2-CBA and second next to 2-CBA also 2,3-CBA and 2,5-CBA. Both groups use for degradation 1,2-dioxygenase reaction. In case of 2-CBA this reaction leads to the formation of catechol (Figure 3.). 2,4-CBA and 2,5-CBA are degraded into 4-chlorocatechol (Figure 4.) [35].

For the 4-chlorocatechol degradation formed in 1,2-dioxygenase reaction with 2,4-CBA or 2,5- CBA it is necessary to have chlorocatechol degradation pathway. It is therefore assumed that the difference between both groups of microorganisms is given by presence of the chloroca‐ techol degradation pathway [9] despite the fact that two different 2-halobenzoate-1,2-dioxy‐ genases are known.

*2.2.1. Degradation of 2-chlorobenzoic acid*

4 Applied Bioremediation - Active and Passive Approaches

COOH

2-chlorobenzoic acid

Cl

reactions proceed only in a small degree [9].

4-chlorocatechol (Figure 4.) [35].

genases are known.

1,2 activity

1,6 activity

2,3 activity

(Figure 3.).

In case of 2-CBA degradation there are known three different ways of degradation. All of them are about dioxygenation reaction catalyzed by 2-halobenzoate-1,2-dioxygenase (EC 1.14.12.13)

HOOC

HOOC

H

**Figure 3.** Aerobic degradation of 2-chlorobenzoic acid by the enzyme 2-chlorobenzoate-1,2-dioxygenase [9, 38].

Main activity of 2-halobenzoate-1,2-dioxygenase is 1,2-dioxygenase reaction. In this reaction is 2-CBA degraded to catechol. During this reaction are released carbon dioxide and chlorine. Enzyme 2-halobenzoate-1,2-dioxygenase has also 1,6-dioxygenase activity. In 1,6-dioxygenase reaction is formed 3-chlorocatechol. Last activity of 2-halobenzoate-1,2-dioxygenase is 2,3 dioxygenase reaction leading to the formation of 2,3-dihydroxybenzoic acid. Latter two

Degradation of 2-CBA by 2-halobenzoate-1,2-dioxygenase was reported in two groups of microorganisms. First group degraded only 2-CBA and second next to 2-CBA also 2,3-CBA and 2,5-CBA. Both groups use for degradation 1,2-dioxygenase reaction. In case of 2-CBA this reaction leads to the formation of catechol (Figure 3.). 2,4-CBA and 2,5-CBA are degraded into

For the 4-chlorocatechol degradation formed in 1,2-dioxygenase reaction with 2,4-CBA or 2,5- CBA it is necessary to have chlorocatechol degradation pathway. It is therefore assumed that the difference between both groups of microorganisms is given by presence of the chloroca‐ techol degradation pathway [9] despite the fact that two different 2-halobenzoate-1,2-dioxy‐

OH

OH

Cl

OH

OH

OH

OH

catechol

COOH

3-chlorocatechol

2,3-dihydroxybenzoic acid

OH

Cl

OH

Cl

OH <sup>H</sup>

OH

OH

OH Cl

COOH

**Figure 4.** Degradation of 2,4-dichlorobenzoic and 2,5-dichlorobenzoic acid catalyzed by 2-halobenzoate-1,2-dioxyge‐ nase.

First 2-halobenzoate-1,2-dioxygenase is a two componential enzymatic system [39]. This 2 halobenzoate-1,2-dioxygenase has high homology with the toluate and benzoate-1,2-dioxy‐ genase. Two component 2-halobenzoate-1,2-dioxygenase has high affinity to 2-CBA but low to 4-CBA; 2,4-CBA and 2,5-CBA[9, 40].

Second 2-halobenzoate-1,2-dioxygenase is three componential [35] and this enzyme catalyzes degradation of 2-CBA; 2,4-CBA and 2,5-CBA.

Next to 1,2-dioxygenase activity the enzyme 2-halobenzoate-1,2-dioxygenase has also 1,6 dioxygenase activity [9, 41]. Strain *Pseudomonas* sp. B-300 was in presence of glucose able to degrade 2-CBA to the 3-chlorocatechol [41].Whereas *Pseudomonas* sp. B-300 was cultivated with 2-CBA as the only carbon and energy source just catechol was identified. From that it is obvious that 1,6-dioxygenase reaction occurs only in much smaller degree than 1,2-dioxygenase reaction or in the presence of a rich source of energy.

Dioxygenase reaction in the 1,6- position is followed by 3-chlorocatechol ring cleavage forming chloromuconic acid which is dehalogenated.

Another metabolite identified in medium after cultivation of strain *Burkholderia cepacia* 2CBS with 2-CBA was 2,3-dihydroxybenzoic acid [42]. Strain *Burkholderia cepacia* 2CBS degraded most of 2-CBA by 1,2-dioxygenase reaction, 2,3-dihydroxybenzoic acid was in medium accumulated just in small quantities. Formation of 2,3-dihydroxybenzoic acid indicates that two-component 2-halobenzoate-1,2-dioxygenase of strain *Burkholderia cepacia* 2CBS has next to 1,2-dioxygenase activity also 2,3-dioxygenase activity (Figure 3.). 2,3-dihydroxybenzoic acid is the dead-end product [9].

#### *2.2.2. Degradation of 3-chlorobenzoic acid*

Degradation of 3-CBA can be done by several different degradation pathways. Benzoate-1,2 dioxygenase catalyzes the conversion of 3-CBA into 3-chlorocatechol or 4-chlorocatechol. 3- CBA can be also transformed to the protocatechuate (3,4-dihydroxybenzoic acid) or 5 chloroprotocatechuate (5-chloro-3,4-dihydroxybenzoic acid) by the enzyme 3 chlorobenzoate-4,5-dioxygenase. Another possibility is the degradation of 3-CBA via 3 hydroxybenzoic acid to the gentisic acid (2,5-dihydroxybenzoic acid).

The enzyme 3-chlorobenzoate-3,4-dioxygenase can catalyze also degradation of 3,4-CBA. 3,4- CBA is degraded to the 5-chloroprotocatechuic acid [34, 48] which means that 3,4-CBA is

Cl

HO

COOH

<sup>H</sup> HO

**Figure 6.** Degradation pathway of 3-chlorobenzoic and 3,4-dichlorobenzoic acid by 3-chlorobenzoate-3,4-dioxyge‐

Cl

3-CBA can be also degraded by monooxygenase reaction. This reaction leads to the gentisic acid with 3-hydroxybenzoic acid as an intermediate (Figure 7.) [37]. The enzyme for the conversion of 3-CBA to the 3-hydroxybenzoic acid is not yet known, the second reaction is

Figure 6. Degradation pathway of 3-chlorobenzoic and 3,4-dichlorobenzoic acid by 3-chlorobenzoate-3,4-dioxygenase.

dioxygenase 3-chlorobenzoate-4,5-

COOH

Cl

HO

COOH

OH

acid

protokatechuic

OH

OH

HO

acid

dioxygenase 3-chlorobenzoate-4,5-

5-chloroprotokatechuic

COOH

Cl

COOH

OH

acid

3-CBA can be also degraded by monooxygenase reaction. This reaction leads to the gentisic acid with 3-hydroxybenzoic acid as an intermediate (Figure 7.) [37]. The enzyme for the conversion of 3-CBA to the 3-hydroxybenzoic acid is not yet known, the second

Monooxygenase reaction was reported in strains *Pseudomonas* sp. [50] and *Alcaligenes* sp. L6 [37]. Strain *Alcaligenes* sp. L6 has next to this pathway also pathway for 3-CBA degradation with protocatechuic acid as the final product. Strain L6 was isolated under low oxygen concentration. From this it can be assumed that 3-CBA degradation by monooxygenase reaction is held only under low oxygen concentration. When such conditions do not occur, the preferred microorganisms are those which can 3-CBA degrade by

NADH+H+ NAD

H2O

Microorganisms able of the degradation of 4-CBA mostly belong to the strains *Alcaligenes, Arthrobacter* and *Pseudomonas* [51]. Until now, two pathways for 4-CBA degradation were described. In first, the more common, is 4-CBA dehalogenation followed by ring cleavage. In the second is 4-CBA converted to 4-chlorocatechol which is further subjected to the ring cleavage and only then is

In first mentioned 4-CBA degradation pathway is 4-hydroxybenzoic acid formed (Figure 8.). This pathway begins with the conversion of 4-CBA to 4-chlorobenzoate-CoA catalyzed by 4-chlorobenzoate:CoA ligase with the consumption of 1 molecule of ATP [53]. This reaction is followed by replacement of chlorine atom with hydroxyl group derived from water catalyzed by 4 chlorobenzoate:CoA dehalogenase [54]. The last step of 4-CBA dehalogenation is the hydrolysis of 4-hydroxybenzoate-CoA

thioester by the enzyme 4-hydroxybenzoate:CoA thioesterase with formation of 4-hydroxybenzoate [32].

dioxygenase reaction. It may be also the reason why are described only a few strains degrading 3-CBA via gentisic acid.

O2

protokatechuic

COOH

4,5 activity

dioxygenase

Cl

H

COOH

HO

Cl

Cl HO

H

Bioremediation of Chlorobenzoic Acids http://dx.doi.org/10.5772/56394

COOH

dioxygenase

OH

acid

5-chloroprotokatechuic

acid

OH

HO

3,4-dichlorobenzoic

Cl

COOH

gentisic acid

OH

Cl

acid

3,4-dichlorobenzoic

HO

Cl HO

7

COOH

4,5 activity

Cl

COOH

Cl

Cl

COOH

H

HO

Cl

OH

H OH

COOH

OH

H OH

catalyzed by 3-hydroxybenzoate-6-hydroxylase (EC 1.14.13.24) [49].

reaction is catalyzed by 3-hydroxybenzoate-6-hydroxylase (EC 1.14.13.24) [49].

COOH

Figure 7. Degradation pathway of 3-chlorobenzoic acid with gentisic acid as an intermediate.

3-hydroxybenzoic acid

**Figure 7.** Degradation pathway of 3-chlorobenzoic acid with gentisic acid as an intermediate.

OH

Monooxygenase reaction was reported in strains *Pseudomonas* sp. [50] and *Alcaligenes* sp. L6 [37]. Strain *Alcaligenes* sp. L6 has next to this pathway also pathway for 3-CBA degradation with protocatechuic acid as the final product. Strain L6 was isolated under low oxygen concentration. From this it can be assumed that 3-CBA degradation by monooxygenase

? 3-hydroxybenzoate-6-hydroxylase

**2.2.3. Degradation of 4-chlorobenzoic acid** 

dehalogenated [52].

<sup>H</sup> HO

H

degraded by the 4,5-dioxygenase reaction.

3-chlorbenzoate-4,5-

Cl

3,4 activity

4,5 activity

3-chlorbenzoate-4,5-

3,4 activity

4,5 activity

COOH

acid

nase.

3-chlorobenzoic

Cl

acid

COOH

3-chlorobenzoic acid

Cl

H2O HCl

3-chlorobenzoic

COOH

First mentioned 3-CBA degradation is degradation in the benzoate degradation pathway. Breakdown of 3-CBA is catalyzed by the enzyme benzoate-1,2-dioxygenase (EC 1.14.12.10). Benzoate-1,2-dioxygenase is a wide spread enzyme which has been identified in many microorganisms. Substrate specificity of this enzyme is relatively narrow. Benzoate-1,2 dioxygenasa catalyzes only conversion of benzoate, 3-CBA and 3-methylbenzoate [9, 43, 44].

The mechanism of reaction catalyzed by the benzoate-1,2-dioxygenase is based on the double hydroxylation in 1,2- or 1,6-position of benzene ring. The final product of this reaction is catechol in case of benzoic acid or 4-chlorocatechol or 3-chlorocatechol in case of 3-CBA degradation (Figure 5.).

Benzoate-1,2-dioxygenase of the strain *Alcaligenes eutrophus* JMP134 [43] form from 3-CBA 3 chlorocatechol and 4-chlorocatechol in a 1:2 ratio, as well as the benzoate-1,2-dioxygenase of strain *Pseudomonas* sp. B13 [44] do.

**Figure 5.** Degradation pathway for 3-chlorobenzoic acid by benzoate-1,2-dioxygenase [9] with indicated 1,2-dioxyge‐ nase and 1,6-dioxygenase activity.

Next to benzoate-1,2-dioxygenase 3-CBA can be degraded also by the 3-chlorobenzoate-3,4 dioxygenase [45-47]. Main activity of this enzyme is 4,5-dioxygenase reaction with formation of 5-chloroprotocatechuic acid. Besides the 4,5-dioxygenase reaction is a small amount of 3- CBA transformed by 3,4-dioxygenase reaction to the protocatechuic acid (Figure 6.).

COOH

4,5 activity

Cl

COOH

HO

acid

Cl

Cl

The enzyme 3-chlorobenzoate-3,4-dioxygenase can catalyze also degradation of 3,4-CBA. 3,4- CBA is degraded to the 5-chloroprotocatechuic acid [34, 48] which means that 3,4-CBA is degraded by the 4,5-dioxygenase reaction.

*2.2.2. Degradation of 3-chlorobenzoic acid*

6 Applied Bioremediation - Active and Passive Approaches

degradation (Figure 5.).

COOH

3-chlorobenzoic acid

nase and 1,6-dioxygenase activity.

strain *Pseudomonas* sp. B13 [44] do.

Degradation of 3-CBA can be done by several different degradation pathways. Benzoate-1,2 dioxygenase catalyzes the conversion of 3-CBA into 3-chlorocatechol or 4-chlorocatechol. 3- CBA can be also transformed to the protocatechuate (3,4-dihydroxybenzoic acid) or 5 chloroprotocatechuate (5-chloro-3,4-dihydroxybenzoic acid) by the enzyme 3 chlorobenzoate-4,5-dioxygenase. Another possibility is the degradation of 3-CBA via 3-

First mentioned 3-CBA degradation is degradation in the benzoate degradation pathway. Breakdown of 3-CBA is catalyzed by the enzyme benzoate-1,2-dioxygenase (EC 1.14.12.10). Benzoate-1,2-dioxygenase is a wide spread enzyme which has been identified in many microorganisms. Substrate specificity of this enzyme is relatively narrow. Benzoate-1,2 dioxygenasa catalyzes only conversion of benzoate, 3-CBA and 3-methylbenzoate [9, 43, 44].

The mechanism of reaction catalyzed by the benzoate-1,2-dioxygenase is based on the double hydroxylation in 1,2- or 1,6-position of benzene ring. The final product of this reaction is catechol in case of benzoic acid or 4-chlorocatechol or 3-chlorocatechol in case of 3-CBA

Benzoate-1,2-dioxygenase of the strain *Alcaligenes eutrophus* JMP134 [43] form from 3-CBA 3 chlorocatechol and 4-chlorocatechol in a 1:2 ratio, as well as the benzoate-1,2-dioxygenase of

Cl OH

**Figure 5.** Degradation pathway for 3-chlorobenzoic acid by benzoate-1,2-dioxygenase [9] with indicated 1,2-dioxyge‐

Next to benzoate-1,2-dioxygenase 3-CBA can be degraded also by the 3-chlorobenzoate-3,4 dioxygenase [45-47]. Main activity of this enzyme is 4,5-dioxygenase reaction with formation of 5-chloroprotocatechuic acid. Besides the 4,5-dioxygenase reaction is a small amount of 3-

CBA transformed by 3,4-dioxygenase reaction to the protocatechuic acid (Figure 6.).

1,2 activity

1,6 activity

HOOC

benzoate-1,2-dioxygenase 3-chlorocatechol

OH <sup>H</sup>HO COOH

OH

Cl

Cl

H

OH

HO

Cl

Cl

OH

OH

4-chlorocatechol

hydroxybenzoic acid to the gentisic acid (2,5-dihydroxybenzoic acid).

**Figure 6.** Degradation pathway of 3-chlorobenzoic and 3,4-dichlorobenzoic acid by 3-chlorobenzoate-3,4-dioxyge‐ nase. acid protokatechuic

OH

OH

3-CBA can be also degraded by monooxygenase reaction. This reaction leads to the gentisic acid with 3-hydroxybenzoic acid as an intermediate (Figure 7.) [37]. The enzyme for the conversion of 3-CBA to the 3-hydroxybenzoic acid is not yet known, the second reaction is catalyzed by 3-hydroxybenzoate-6-hydroxylase (EC 1.14.13.24) [49]. Figure 6. Degradation pathway of 3-chlorobenzoic and 3,4-dichlorobenzoic acid by 3-chlorobenzoate-3,4-dioxygenase. 3-CBA can be also degraded by monooxygenase reaction. This reaction leads to the gentisic acid with 3-hydroxybenzoic acid as an intermediate (Figure 7.) [37]. The enzyme for the conversion of 3-CBA to the 3-hydroxybenzoic acid is not yet known, the second

Figure 7. Degradation pathway of 3-chlorobenzoic acid with gentisic acid as an intermediate. **Figure 7.** Degradation pathway of 3-chlorobenzoic acid with gentisic acid as an intermediate.

**2.2.3. Degradation of 4-chlorobenzoic acid** 

dehalogenated [52].

reaction is catalyzed by 3-hydroxybenzoate-6-hydroxylase (EC 1.14.13.24) [49].

Monooxygenase reaction was reported in strains *Pseudomonas* sp. [50] and *Alcaligenes* sp. L6 [37]. Strain *Alcaligenes* sp. L6 has next to this pathway also pathway for 3-CBA degradation with protocatechuic acid as the final product. Strain L6 was isolated under low oxygen concentration. From this it can be assumed that 3-CBA degradation by monooxygenase reaction is held only under low oxygen concentration. When such conditions do not occur, the preferred microorganisms are those which can 3-CBA degrade by dioxygenase reaction. It may be also the reason why are described only a few strains degrading 3-CBA via gentisic acid. Monooxygenase reaction was reported in strains *Pseudomonas* sp. [50] and *Alcaligenes* sp. L6 [37]. Strain *Alcaligenes* sp. L6 has next to this pathway also pathway for 3-CBA degradation with protocatechuic acid as the final product. Strain L6 was isolated under low oxygen concentration. From this it can be assumed that 3-CBA degradation by monooxygenase

thioester by the enzyme 4-hydroxybenzoate:CoA thioesterase with formation of 4-hydroxybenzoate [32].

Microorganisms able of the degradation of 4-CBA mostly belong to the strains *Alcaligenes, Arthrobacter* and *Pseudomonas* [51]. Until now, two pathways for 4-CBA degradation were described. In first, the more common, is 4-CBA dehalogenation followed by ring cleavage. In the second is 4-CBA converted to 4-chlorocatechol which is further subjected to the ring cleavage and only then is

In first mentioned 4-CBA degradation pathway is 4-hydroxybenzoic acid formed (Figure 8.). This pathway begins with the conversion of 4-CBA to 4-chlorobenzoate-CoA catalyzed by 4-chlorobenzoate:CoA ligase with the consumption of 1 molecule of ATP [53]. This reaction is followed by replacement of chlorine atom with hydroxyl group derived from water catalyzed by 4 chlorobenzoate:CoA dehalogenase [54]. The last step of 4-CBA dehalogenation is the hydrolysis of 4-hydroxybenzoate-CoA reaction is held only under low oxygen concentration. When such conditions do not occur, the preferred microorganisms are those which can 3-CBA degrade by dioxygenase reaction. It may be also the reason why are described only a few strains degrading 3-CBA via gentisic acid.

Temporary formation of CoA thioester, one step after it was created, is special due to the energy consuming production of thioester bond. At the same time thioester formation is not required

Genes coding this operon can be located in the chromosomal DNA as well as on a plasmid [32].

CO-SCoA

H2O

*I II III*

HCl

Cl

chlorobenzoate:CoA dehalogenase; *III* - 4-hydroxybenzoate:CoA thioesterase

AMP + PPi H2O

Figure 8. Degradation pathway of 4-chlorobenzoic acid with formation of 4-hydroxybenzoic acid. *I* – 4-chlorobenzoate:CoA ligase; *II* - 4-

OH

4-chlorobenzoate-CoA 4-hydroxybenzoic

4-hydroxybenzoate-CoA

CO-SCoA

H2O

HSCoA + H<sup>+</sup>

All of the enzymes required for this conversion are organized in one operon. This operon is regulated by the presence of 4-CBA.

The order of genes in the operon is for each bacterial strain different. *Pseudomonas* strain has dehalogenase-ligase-thioesterase and strain *Arthrobacter* has ligase-dehalogenase-thioesterase [32]. This indicates that the genes coding dehalogenation of 4-CBA have been rearranged. The low agreement of protein sequences indicates that this pathway is not the result of a recent adaptation. Therefore it is not the result of the recent release of PCBs and thus also 4-CBA, but it can be assumed that the 4-CBA dehalogenation pathway had enough time to arise from random mutations and selections. Indirectly it can be assumed that there is

Temporary formation of CoA thioester, one step after it was created, is special due to the energy consuming production of thioester

In case of anaerobic degradation of 3-CBA by strain *Rhodopseudomonas palustris* RCB100 is 3-chlorobenzoate-CoA formed. Its formation is followed by dehalogenation with release of benzoate-CoA. Benzoate-CoA is further degraded to the acetyl-CoA [56]. Same pathway is used for benzoic acid degradation. This pathway begins with formation of benzoate-CoA and CoA remains bound through the whole degradation pathway [24]. For the aerobic degradation of 4-CBA is thioester required only for the efficient dehalogenation. Thus, consumption of one molecule of ATP is acceptable due to the fact that it allows degradation of the

The second possibility of 4-CBA degradation is degradation with 4-chlorocatechol as an intermediate (Figure 9.). Formation of 4 chlorocatechol is followed by ring cleavage and dehalogenation. This way of 4-CBA degradation is much less investigated than 4-

> OH <sup>H</sup>HO COOH

> > Cl

Enzyme of first step of 4-CBA degradation to the 4-chlorocatechol can be either the toluate-1,2-dioxygenase (EC 1.14.12.10) [31] like in case of strain *Pseudomonas aeruginosa* mt-2 or by not yet closer identified chlorobenzoate-1,2-dioxygenase described for example

Strain *Pseudomonas aeruginosa* 3mT was also able to degrade 3-CBA with formation of 3-chlorocatechol but formation of 4 chlorocatechol was not recorded. This activity suggest that *Pseudomonas aeruginosa* 3mT dioxygenase has only 1,2-dioxygenase activity. Not like benzoate-1,2-dioxygenase and 2-halobenzoate-1,2-dioxygenase 1,2- and 1,6-dioxygenase activity. From this can be

assumed that dioxygenase from strain *Pseudomonas aeruginosa* 3mT is not identical with either of these enzymes.

4-chlorobenzoic 4-chlorocatechol

Enzyme of first step of 4-CBA degradation to the 4-chlorocatechol can be either the toluate-1,2 dioxygenase (EC 1.14.12.10) [31] like in case of strain *Pseudomonas aeruginosa* mt-2 or by not yet closer identified chlorobenzoate-1,2-dioxygenase described for example in the strain *Pseudo‐*

Strain *Pseudomonas aeruginosa* 3mT was also able to degrade 3-CBA with formation of 3 chlorocatechol but formation of 4-chlorocatechol was not recorded. This activity suggest that *Pseudomonas aeruginosa* 3mT dioxygenase has only 1,2-dioxygenase activity. Not like ben‐ zoate-1,2-dioxygenase and 2-halobenzoate-1,2-dioxygenase 1,2- and 1,6-dioxygenase activity. From this can be assumed that dioxygenase from strain *Pseudomonas aeruginosa* 3mT is not

Aerobic degradation of CBA with two chlorine atoms in molecule has been reported for many strains [17]. Degradation of CBA with three chlorine atoms is relatively rare, in the literature

OH

COOH

OH

acid

9

Bioremediation of Chlorobenzoic Acids http://dx.doi.org/10.5772/56394

Cl

HO

In case of anaerobic degradation of 3-CBA by strain *Rhodopseudomonas palustris* RCB100 is 3 chlorobenzoate-CoA formed. Its formation is followed by dehalogenation with release of benzoate-CoA. Benzoate-CoA is further degraded to the acetyl-CoA [56]. Same pathway is used for benzoic acid degradation. This pathway begins with formation of benzoate-CoA and CoA remains bound through the whole degradation pathway [24]. For the aerobic degradation of 4-CBA is thioester required only for the efficient dehalogenation. Thus, consumption of one molecule of ATP is acceptable due to the fact that it allows degradation of the molecule and

The second possibility of 4-CBA degradation is degradation with 4-chlorocatechol as an intermediate (Figure 9.). Formation of 4-chlorocatechol is followed by ring cleavage and dehalogenation. This way of 4-CBA degradation is much less investigated than 4-CBA

bond. At the same time thioester formation is not required for aerobic degradation of other CBA.

for aerobic degradation of other CBAs.

COOH

Cl

4-chlorobenzoic acid

ATP + CoASH + H<sup>+</sup>

a natural source of 4-CBA [55].

COOH

Cl

acid

*monas aeruginosa* 3mT [36].

identical with either of these enzymes.

enables its use as a source of carbon and energy.

degradation leading to the formation of 4-hydroxybenzoate.

toluate-1,2-dioxygenase

in the strain *Pseudomonas aeruginosa* 3mT [36].

*2.2.4. Degradation of more chlorinated chlorobenzoic acids*

Figure 9. Degradation pathway of 4-chlorobenzoic acid leading to the 4-chlorocatechol

**Figure 9.** Degradation pathway of 4-chlorobenzoic acid leading to the 4-chlorocatechol

**2.2.4. Degradation of more chlorinated chlorobenzoic acids** 

molecule and enables its use as a source of carbon and energy.

CBA degradation leading to the formation of 4-hydroxybenzoate.

#### *2.2.3. Degradation of 4-chlorobenzoic acid*

Microorganisms able of the degradation of 4-CBA mostly belong to the strains *Alcaligenes, Arthrobacter* and *Pseudomonas* [51]. Until now, two pathways for 4-CBA degradation were described. In first, the more common, is 4-CBA dehalogenation followed by ring cleavage. In the second is 4-CBA converted to 4-chlorocatechol which is further subjected to the ring cleavage and only then is dehalogenated [52].

In first mentioned 4-CBA degradation pathway is 4-hydroxybenzoic acid formed (Figure 8.). This pathway begins with the conversion of 4-CBA to 4-chlorobenzoate-CoA catalyzed by 4 chlorobenzoate:CoA ligase with the consumption of 1 molecule of ATP [53]. This reaction is followed by replacement of chlorine atom with hydroxyl group derived from water catalyzed by 4-chlorobenzoate:CoA dehalogenase [54]. The last step of 4-CBA dehalogenation is the hydrolysis of 4-hydroxybenzoate-CoA thioester by the enzyme 4-hydroxybenzoate:CoA thioesterase with formation of 4-hydroxybenzoate [32].

Figure 8. Degradation pathway of 4-chlorobenzoic acid with formation of 4-hydroxybenzoic acid. *I* – 4-chlorobenzoate:CoA ligase; *II* - 4 chlorobenzoate:CoA dehalogenase; *III* - 4-hydroxybenzoate:CoA thioesterase **Figure 8.** Degradation pathway of 4-chlorobenzoic acid with formation of 4-hydroxybenzoic acid. *I* – 4-chloroben‐ zoate:CoA ligase; *II* - 4-chlorobenzoate:CoA dehalogenase; *III* - 4-hydroxybenzoate:CoA thioesterase

All of the enzymes required for this conversion are organized in one operon. This operon is regulated by the presence of 4-CBA. Genes coding this operon can be located in the chromosomal DNA as well as on a plasmid [32]. The order of genes in the operon is for each bacterial strain different. *Pseudomonas* strain has dehalogenase-ligase-thioesterase and All of the enzymes required for this conversion are organized in one operon. This operon is regulated by the presence of 4-CBA. Genes coding this operon can be located in the chromo‐ somal DNA as well as on a plasmid [32].

strain *Arthrobacter* has ligase-dehalogenase-thioesterase [32]. This indicates that the genes coding dehalogenation of 4-CBA have

bound through the whole degradation pathway [24]. For the aerobic degradation of 4-CBA is thioester required only for the efficient dehalogenation. Thus, consumption of one molecule of ATP is acceptable due to the fact that it allows degradation of the

The second possibility of 4-CBA degradation is degradation with 4-chlorocatechol as an intermediate (Figure 9.). Formation of 4 chlorocatechol is followed by ring cleavage and dehalogenation. This way of 4-CBA degradation is much less investigated than 4-

> OH <sup>H</sup>HO COOH

> > Cl

Enzyme of first step of 4-CBA degradation to the 4-chlorocatechol can be either the toluate-1,2-dioxygenase (EC 1.14.12.10) [31] like in case of strain *Pseudomonas aeruginosa* mt-2 or by not yet closer identified chlorobenzoate-1,2-dioxygenase described for example

Strain *Pseudomonas aeruginosa* 3mT was also able to degrade 3-CBA with formation of 3-chlorocatechol but formation of 4 chlorocatechol was not recorded. This activity suggest that *Pseudomonas aeruginosa* 3mT dioxygenase has only 1,2-dioxygenase activity. Not like benzoate-1,2-dioxygenase and 2-halobenzoate-1,2-dioxygenase 1,2- and 1,6-dioxygenase activity. From this can be

assumed that dioxygenase from strain *Pseudomonas aeruginosa* 3mT is not identical with either of these enzymes.

4-chlorobenzoic 4-chlorocatechol

OH

Cl

HO

been rearranged. The low agreement of protein sequences indicates that this pathway is not the result of a recent adaptation. Therefore it is not the result of the recent release of PCBs and thus also 4-CBA, but it can be assumed that the 4-CBA dehalogenation pathway had enough time to arise from random mutations and selections. Indirectly it can be assumed that there is a natural source of 4-CBA [55]. Temporary formation of CoA thioester, one step after it was created, is special due to the energy consuming production of thioester bond. At the same time thioester formation is not required for aerobic degradation of other CBA. In case of anaerobic degradation of 3-CBA by strain *Rhodopseudomonas palustris* RCB100 is 3-chlorobenzoate-CoA formed. Its formation is followed by dehalogenation with release of benzoate-CoA. Benzoate-CoA is further degraded to the acetyl-CoA [56]. Same pathway is used for benzoic acid degradation. This pathway begins with formation of benzoate-CoA and CoA remains The order of genes in the operon is for each bacterial strain different. *Pseudomonas* strain has dehalogenase-ligase-thioesterase and strain *Arthrobacter* has ligase-dehalogenase-thioesterase [32]. This indicates that the genes coding dehalogenation of 4-CBA have been rearranged. The low agreement of protein sequences indicates that this pathway is not the result of a recent adaptation. Therefore it is not the result of the recent release of PCBs and thus also 4-CBA, but it can be assumed that the 4-CBA dehalogenation pathway had enough time to arise from random mutations and selections. Indirectly it can be assumed that there is a natural source of 4-CBA [55].

molecule and enables its use as a source of carbon and energy.

CBA degradation leading to the formation of 4-hydroxybenzoate.

toluate-1,2-dioxygenase

in the strain *Pseudomonas aeruginosa* 3mT [36].

Figure 9. Degradation pathway of 4-chlorobenzoic acid leading to the 4-chlorocatechol

**2.2.4. Degradation of more chlorinated chlorobenzoic acids** 

COOH

Cl

acid

OH

COOH

Temporary formation of CoA thioester, one step after it was created, is special due to the energy consuming production of thioester bond. At the same time thioester formation is not required for aerobic degradation of other CBAs. All of the enzymes required for this conversion are organized in one operon. This operon is regulated by the presence of 4-CBA. Genes coding this operon can be located in the chromosomal DNA as well as on a plasmid [32]. The order of genes in the operon is for each bacterial strain different. *Pseudomonas* strain has dehalogenase-ligase-thioesterase and

CO-SCoA

H2O

*I II III*

HCl

Cl

chlorobenzoate:CoA dehalogenase; *III* - 4-hydroxybenzoate:CoA thioesterase

AMP + PPi H2O

COOH

Cl

4-chlorobenzoic acid

ATP + CoASH + H<sup>+</sup>

reaction is held only under low oxygen concentration. When such conditions do not occur, the preferred microorganisms are those which can 3-CBA degrade by dioxygenase reaction. It may be also the reason why are described only a few strains degrading 3-CBA via gentisic acid.

Microorganisms able of the degradation of 4-CBA mostly belong to the strains *Alcaligenes, Arthrobacter* and *Pseudomonas* [51]. Until now, two pathways for 4-CBA degradation were described. In first, the more common, is 4-CBA dehalogenation followed by ring cleavage. In the second is 4-CBA converted to 4-chlorocatechol which is further subjected to the ring

In first mentioned 4-CBA degradation pathway is 4-hydroxybenzoic acid formed (Figure 8.). This pathway begins with the conversion of 4-CBA to 4-chlorobenzoate-CoA catalyzed by 4 chlorobenzoate:CoA ligase with the consumption of 1 molecule of ATP [53]. This reaction is followed by replacement of chlorine atom with hydroxyl group derived from water catalyzed by 4-chlorobenzoate:CoA dehalogenase [54]. The last step of 4-CBA dehalogenation is the hydrolysis of 4-hydroxybenzoate-CoA thioester by the enzyme 4-hydroxybenzoate:CoA

Figure 8. Degradation pathway of 4-chlorobenzoic acid with formation of 4-hydroxybenzoic acid. *I* – 4-chlorobenzoate:CoA ligase; *II* - 4-

OH

4-chlorobenzoate-CoA 4-hydroxybenzoic

4-hydroxybenzoate-CoA

CO-SCoA

H2O

HSCoA + H<sup>+</sup>

All of the enzymes required for this conversion are organized in one operon. This operon is regulated by the presence of 4-CBA.

The order of genes in the operon is for each bacterial strain different. *Pseudomonas* strain has dehalogenase-ligase-thioesterase and strain *Arthrobacter* has ligase-dehalogenase-thioesterase [32]. This indicates that the genes coding dehalogenation of 4-CBA have been rearranged. The low agreement of protein sequences indicates that this pathway is not the result of a recent adaptation. Therefore it is not the result of the recent release of PCBs and thus also 4-CBA, but it can be assumed that the 4-CBA dehalogenation pathway had enough time to arise from random mutations and selections. Indirectly it can be assumed that there is

Temporary formation of CoA thioester, one step after it was created, is special due to the energy consuming production of thioester

In case of anaerobic degradation of 3-CBA by strain *Rhodopseudomonas palustris* RCB100 is 3-chlorobenzoate-CoA formed. Its formation is followed by dehalogenation with release of benzoate-CoA. Benzoate-CoA is further degraded to the acetyl-CoA [56]. Same pathway is used for benzoic acid degradation. This pathway begins with formation of benzoate-CoA and CoA remains bound through the whole degradation pathway [24]. For the aerobic degradation of 4-CBA is thioester required only for the efficient dehalogenation. Thus, consumption of one molecule of ATP is acceptable due to the fact that it allows degradation of the

The second possibility of 4-CBA degradation is degradation with 4-chlorocatechol as an intermediate (Figure 9.). Formation of 4 chlorocatechol is followed by ring cleavage and dehalogenation. This way of 4-CBA degradation is much less investigated than 4-

> OH <sup>H</sup>HO COOH

> > Cl

Enzyme of first step of 4-CBA degradation to the 4-chlorocatechol can be either the toluate-1,2-dioxygenase (EC 1.14.12.10) [31] like in case of strain *Pseudomonas aeruginosa* mt-2 or by not yet closer identified chlorobenzoate-1,2-dioxygenase described for example

Strain *Pseudomonas aeruginosa* 3mT was also able to degrade 3-CBA with formation of 3-chlorocatechol but formation of 4 chlorocatechol was not recorded. This activity suggest that *Pseudomonas aeruginosa* 3mT dioxygenase has only 1,2-dioxygenase activity. Not like benzoate-1,2-dioxygenase and 2-halobenzoate-1,2-dioxygenase 1,2- and 1,6-dioxygenase activity. From this can be

assumed that dioxygenase from strain *Pseudomonas aeruginosa* 3mT is not identical with either of these enzymes.

4-chlorobenzoic 4-chlorocatechol

OH

COOH

OH

acid

Cl

HO

*2.2.3. Degradation of 4-chlorobenzoic acid*

8 Applied Bioremediation - Active and Passive Approaches

cleavage and only then is dehalogenated [52].

thioesterase with formation of 4-hydroxybenzoate [32].

AMP + PPi H2O

chlorobenzoate:CoA dehalogenase; *III* - 4-hydroxybenzoate:CoA thioesterase

molecule and enables its use as a source of carbon and energy.

CBA degradation leading to the formation of 4-hydroxybenzoate.

toluate-1,2-dioxygenase

in the strain *Pseudomonas aeruginosa* 3mT [36].

Figure 9. Degradation pathway of 4-chlorobenzoic acid leading to the 4-chlorocatechol

**2.2.4. Degradation of more chlorinated chlorobenzoic acids** 

a natural source of 4-CBA [55].

somal DNA as well as on a plasmid [32].

COOH

Cl

4-chlorobenzoic acid

ATP + CoASH + H<sup>+</sup>

COOH

of 4-CBA [55].

Cl

acid

Genes coding this operon can be located in the chromosomal DNA as well as on a plasmid [32].

**Figure 8.** Degradation pathway of 4-chlorobenzoic acid with formation of 4-hydroxybenzoic acid. *I* – 4-chloroben‐

All of the enzymes required for this conversion are organized in one operon. This operon is regulated by the presence of 4-CBA. Genes coding this operon can be located in the chromo‐

The order of genes in the operon is for each bacterial strain different. *Pseudomonas* strain has dehalogenase-ligase-thioesterase and strain *Arthrobacter* has ligase-dehalogenase-thioesterase [32]. This indicates that the genes coding dehalogenation of 4-CBA have been rearranged. The low agreement of protein sequences indicates that this pathway is not the result of a recent adaptation. Therefore it is not the result of the recent release of PCBs and thus also 4-CBA, but it can be assumed that the 4-CBA dehalogenation pathway had enough time to arise from random mutations and selections. Indirectly it can be assumed that there is a natural source

CO-SCoA

H2O

*I II III*

HCl

Cl

zoate:CoA ligase; *II* - 4-chlorobenzoate:CoA dehalogenase; *III* - 4-hydroxybenzoate:CoA thioesterase

bond. At the same time thioester formation is not required for aerobic degradation of other CBA.

In case of anaerobic degradation of 3-CBA by strain *Rhodopseudomonas palustris* RCB100 is 3 chlorobenzoate-CoA formed. Its formation is followed by dehalogenation with release of benzoate-CoA. Benzoate-CoA is further degraded to the acetyl-CoA [56]. Same pathway is used for benzoic acid degradation. This pathway begins with formation of benzoate-CoA and CoA remains bound through the whole degradation pathway [24]. For the aerobic degradation of 4-CBA is thioester required only for the efficient dehalogenation. Thus, consumption of one molecule of ATP is acceptable due to the fact that it allows degradation of the molecule and enables its use as a source of carbon and energy. strain *Arthrobacter* has ligase-dehalogenase-thioesterase [32]. This indicates that the genes coding dehalogenation of 4-CBA have been rearranged. The low agreement of protein sequences indicates that this pathway is not the result of a recent adaptation. Therefore it is not the result of the recent release of PCBs and thus also 4-CBA, but it can be assumed that the 4-CBA dehalogenation pathway had enough time to arise from random mutations and selections. Indirectly it can be assumed that there is a natural source of 4-CBA [55]. Temporary formation of CoA thioester, one step after it was created, is special due to the energy consuming production of thioester bond. At the same time thioester formation is not required for aerobic degradation of other CBA. In case of anaerobic degradation of 3-CBA by strain *Rhodopseudomonas palustris* RCB100 is 3-chlorobenzoate-CoA formed. Its formation is followed by dehalogenation with release of benzoate-CoA. Benzoate-CoA is further degraded to the acetyl-CoA [56].

The second possibility of 4-CBA degradation is degradation with 4-chlorocatechol as an intermediate (Figure 9.). Formation of 4-chlorocatechol is followed by ring cleavage and dehalogenation. This way of 4-CBA degradation is much less investigated than 4-CBA degradation leading to the formation of 4-hydroxybenzoate. bound through the whole degradation pathway [24]. For the aerobic degradation of 4-CBA is thioester required only for the efficient dehalogenation. Thus, consumption of one molecule of ATP is acceptable due to the fact that it allows degradation of the molecule and enables its use as a source of carbon and energy. The second possibility of 4-CBA degradation is degradation with 4-chlorocatechol as an intermediate (Figure 9.). Formation of 4-

Same pathway is used for benzoic acid degradation. This pathway begins with formation of benzoate-CoA and CoA remains

CO-SCoA

H2O

HSCoA + H<sup>+</sup>

OH

4-chlorobenzoate-CoA 4-hydroxybenzoic

4-hydroxybenzoate-CoA

chlorocatechol is followed by ring cleavage and dehalogenation. This way of 4-CBA degradation is much less investigated than 4-

Figure 9. Degradation pathway of 4-chlorobenzoic acid leading to the 4-chlorocatechol **Figure 9.** Degradation pathway of 4-chlorobenzoic acid leading to the 4-chlorocatechol

CBA degradation leading to the formation of 4-hydroxybenzoate.

Enzyme of first step of 4-CBA degradation to the 4-chlorocatechol can be either the toluate-1,2-dioxygenase (EC 1.14.12.10) [31] like in case of strain *Pseudomonas aeruginosa* mt-2 or by not yet closer identified chlorobenzoate-1,2-dioxygenase described for example in the strain *Pseudomonas aeruginosa* 3mT [36]. Strain *Pseudomonas aeruginosa* 3mT was also able to degrade 3-CBA with formation of 3-chlorocatechol but formation of 4 chlorocatechol was not recorded. This activity suggest that *Pseudomonas aeruginosa* 3mT dioxygenase has only 1,2-dioxygenase Enzyme of first step of 4-CBA degradation to the 4-chlorocatechol can be either the toluate-1,2 dioxygenase (EC 1.14.12.10) [31] like in case of strain *Pseudomonas aeruginosa* mt-2 or by not yet closer identified chlorobenzoate-1,2-dioxygenase described for example in the strain *Pseudo‐ monas aeruginosa* 3mT [36].

activity. Not like benzoate-1,2-dioxygenase and 2-halobenzoate-1,2-dioxygenase 1,2- and 1,6-dioxygenase activity. From this can be assumed that dioxygenase from strain *Pseudomonas aeruginosa* 3mT is not identical with either of these enzymes. **2.2.4. Degradation of more chlorinated chlorobenzoic acids**  Strain *Pseudomonas aeruginosa* 3mT was also able to degrade 3-CBA with formation of 3 chlorocatechol but formation of 4-chlorocatechol was not recorded. This activity suggest that *Pseudomonas aeruginosa* 3mT dioxygenase has only 1,2-dioxygenase activity. Not like ben‐ zoate-1,2-dioxygenase and 2-halobenzoate-1,2-dioxygenase 1,2- and 1,6-dioxygenase activity. From this can be assumed that dioxygenase from strain *Pseudomonas aeruginosa* 3mT is not identical with either of these enzymes.

#### *2.2.4. Degradation of more chlorinated chlorobenzoic acids*

Aerobic degradation of CBA with two chlorine atoms in molecule has been reported for many strains [17]. Degradation of CBA with three chlorine atoms is relatively rare, in the literature is described only a few strains and in most cases CBAs with three chlorine atoms in molecule are degraded cometabolically.

Same 2m.c consortium can also cometabolically degrade 2,3-CBA via 3-chlorocatechol to the 2-chloromuconic acid with presence of 2-CBA [60]. Strain *Pseudomonas aeruginosa* JB2 [61] degraded 2,3-CBA to the 4-chlorocatechol. Strain *Pseudomonas aeruginosa* JB2 can use also 2-

As it has been said before, CBAs with three chlorine atoms in molecule are converted in most cases cometabolically. Strain *Brevibacterium* sp. converted 2,3,6-CBA by a set of cometabolic steps to carbon dioxide and water [1]. This aerobic degradation proceeds via 2,3,6-trichloro-4 hydroxybenzoic acid or 2,3,6-trichloro-5-hydroxybenzoic acid to the 2,3,5-trichlorophenol or 2,4,5-trichlorophenol and finally to the 3,5-dichlorocatechol, which is than degraded by

Cl Cl

Cl Cl

Bioremediation of Chlorobenzoic Acids http://dx.doi.org/10.5772/56394 11

OH

3,5-dichlorocatechol

HO

OH

Cl Cl

OH

Cl Cl

OH

3,5-dichlorocatechol

OH

Cl Cl

OH

OH

OH

3,5-dichlorocatechol

OH

OH Cl

2,4,5-trichlorophenol

Cl Cl

OH

Strain *Pseudomonas putida* P111 degraded 2,3,5-CBA to the 3,5-dichlorocatechol (Figure 12.) but use different degradation pathway

2,3,5-trichlorophenol

acid 3,5-dichlorocatechol

Cl

Cl Cl

OH

2,3,5-trichlorophenol

acid 3,5-dichlorocatechol

Cl

Cl Cl

HO Cl

2,4,5-trichlorophenol

CBA, 3-CBA, 2,5-CBA and 2,3,5-CBA as the sole carbon and energy source.

COOH Cl Cl

COOH Cl Cl

OH

2,3,6-trichloro-4-hydroxybenzoic

**Figure 11.** Cometabolic degradation of 2,3,6-trichlorobenzoic acid [1].

Figure 11.Cometabolic degradation of 2,3,6-trichlorobenzoic acid [1].

Cl

1,2-dioxygenation followed by dehalogenation [62].

Cl

COOH

Cl Cl

acid

tion [62].

and leads to the tricarboxylic acid cycle.

different position [63].

Cl

OH

2,3,6-trichloro-4-hydroxybenzoic

COOH Cl Cl

HO Cl

2,3,6-trichloro-5-hydroxybenzoic acid

COOH Cl Cl

Strain *Pseudomonas putida* P111 degraded 2,3,5-CBA to the 3,5-dichlorocatechol (Figure 12.) but use different degradation pathway than strain *Brevibacterium* sp., 2,3,5-CBA was degraded by

OH

Cl Cl

Figure 12.Degradation of 2,3,5-trichlorobenzoic acid by strain *Pseudomonas putida* P111 by 1,2-dioxygenase reaction [62].

**Figure 12.** Degradation of 2,3,5-trichlorobenzoic acid by strain *Pseudomonas putida* P111 by 1,2-dioxygenase reac‐

**2.2.5. Degradation of main chlorobenzoic acids degradation intermediates** 

HOOC

2,3,5-trichlorobenzoic CO2

acid, chloroprotocatechuic acid and more chlorinated catechols.

than strain *Brevibacterium* sp., 2,3,5-CBA was degraded by 1,2-dioxygenation followed by dehalogenation [62].

OH

Cl Cl O2 HCl

In CBAs degradation are formed catechol, 3-chlorocatechol, 4-chlorocatechol, 4-hydroxybenzoic acid, gentisic acid, protocatechuic

Catechols can be degraded via *ortho*- or *meta*- cleavage pathway or possibly by modified *ortho-* cleavage pathway, which leads to the 3-oxoadipoic acid pathway. 3-oxoadipoic acid pathway is used for 4-hydroxybenzoic acid and protocatechuc acid degradation

In *ortho*- cleavage pathway is catechol or chlorocatechol degraded by catechol-1,2-dioxygenase or chlorocatechol-1,2-dioxygenase (Figure 13.). The first step of this pathway is dioxygenase reaction leading to the cleavage of the bond between the first and second position of the benzene ring. This reaction produces *cis,cis*-muconic acid or chloro-*cis,cis*-muconic acid with chlorine atom in a

Cl

Cl

OH Cl

2,3,6-trichloro-5-hydroxybenzoic acid

chlorocatechol degradation pathway (Figure 11.).

COOH Cl Cl

2,3,6-trichlorobenzoic acid

Cl

2,3,6-trichlorobenzoic acid

COOH Cl Cl

Dichlorinated CBA can be degraded by a dioxygenase reaction. For example 2,4-CBA and 2,5- CBA which are degraded by three componential 2-halobenzoate-1,2-dioxygenase with release of 4-chlorocatechol, as described in section 2.2.1. Also degradation of 3,4-CBA catalyzed by 3 chlorobenzoate-3,4-dioxygenase with formation of 5-chloroprotocatechuate has been previ‐ ously described in section 2.2.2.

In case of 2,4-CBA is known one more degradation pathway similar to the 4-CBA degradation described in section 2.2.3. In this pathway is 2,4-CBA degraded via 4-hydroxybenzoate to the protocatechuic acid (Figure 10.). This pathway has been described in strains *Corynebacterium sepedonicum* KZ-4 [57] and *Alcaligenes denitrificans* NTB-1 [58].

**Figure 10.** Degradation pathway of 2,4-dichlorobenzoic acid by strain *Corynebacterium sepedonicum* KZ-4 [57]. *I* - 2,4-dichlorobenzoate:CoA ligase; *II* – 2,4-dichlorobenzoate:CoA reductase; *III* – 4-chlorobenzoate:CoA dehalogenase; *IV* – 4-chlorobenzoate:CoA thioesterase; *V* – 4-hydroxybenzoate 3-monooxygenase; TCA – tricarboxylic acid cycle

Strain *Pseudomonas* sp. WR912 [59] can degrade 3,5-CBA by 1,2-dioxygenase reaction with release of 3,5-dichlorocatechol, which was by the same strain degraded to the succinate. Another way of 3,5-CBA degradation is the cometabolic degradation by consortia 2m.c [60]. 3,5-CBA was cometabolically converted to the 3,5-dichlorocatechol as a final intermediate by enzyme 2-halobenzoate-1,2-dioxygenase with presence of 2-CBA and 2,5-CBA. Presence of 3,5- CBA leads to the loss of 2-CBA degradation ability of the 2m.c consortium.

Same 2m.c consortium can also cometabolically degrade 2,3-CBA via 3-chlorocatechol to the 2-chloromuconic acid with presence of 2-CBA [60]. Strain *Pseudomonas aeruginosa* JB2 [61] degraded 2,3-CBA to the 4-chlorocatechol. Strain *Pseudomonas aeruginosa* JB2 can use also 2- CBA, 3-CBA, 2,5-CBA and 2,3,5-CBA as the sole carbon and energy source.

As it has been said before, CBAs with three chlorine atoms in molecule are converted in most cases cometabolically. Strain *Brevibacterium* sp. converted 2,3,6-CBA by a set of cometabolic steps to carbon dioxide and water [1]. This aerobic degradation proceeds via 2,3,6-trichloro-4 hydroxybenzoic acid or 2,3,6-trichloro-5-hydroxybenzoic acid to the 2,3,5-trichlorophenol or 2,4,5-trichlorophenol and finally to the 3,5-dichlorocatechol, which is than degraded by chlorocatechol degradation pathway (Figure 11.).

**Figure 11.** Cometabolic degradation of 2,3,6-trichlorobenzoic acid [1]. OH 2,3,6-trichloro-4-hydroxybenzoic acid 3,5-dichlorocatechol

acid, chloroprotocatechuic acid and more chlorinated catechols.

and leads to the tricarboxylic acid cycle.

different position [63].

is described only a few strains and in most cases CBAs with three chlorine atoms in molecule

Dichlorinated CBA can be degraded by a dioxygenase reaction. For example 2,4-CBA and 2,5- CBA which are degraded by three componential 2-halobenzoate-1,2-dioxygenase with release of 4-chlorocatechol, as described in section 2.2.1. Also degradation of 3,4-CBA catalyzed by 3 chlorobenzoate-3,4-dioxygenase with formation of 5-chloroprotocatechuate has been previ‐

In case of 2,4-CBA is known one more degradation pathway similar to the 4-CBA degradation described in section 2.2.3. In this pathway is 2,4-CBA degraded via 4-hydroxybenzoate to the protocatechuic acid (Figure 10.). This pathway has been described in strains *Corynebacterium*

*sepedonicum* KZ-4 [57] and *Alcaligenes denitrificans* NTB-1 [58].

CoA-SH Cl

COOH

OH

acid

Cl

*I II*

OH

NAD<sup>+</sup> + H2O

CBA leads to the loss of 2-CBA degradation ability of the 2m.c consortium.

SO CoA

NADPH

*V*

3,4-dihydroxybenzoic 4-hydroxybenzoic

NADP+ + Cl-

2,4-dichlorobenzoate-CoA 4-chlorobenzoate-CoA

NADH + O2

**Figure 10.** Degradation pathway of 2,4-dichlorobenzoic acid by strain *Corynebacterium sepedonicum* KZ-4 [57]. *I* - 2,4-dichlorobenzoate:CoA ligase; *II* – 2,4-dichlorobenzoate:CoA reductase; *III* – 4-chlorobenzoate:CoA dehalogenase; *IV* – 4-chlorobenzoate:CoA thioesterase; *V* – 4-hydroxybenzoate 3-monooxygenase; TCA – tricarboxylic acid cycle

Strain *Pseudomonas* sp. WR912 [59] can degrade 3,5-CBA by 1,2-dioxygenase reaction with release of 3,5-dichlorocatechol, which was by the same strain degraded to the succinate. Another way of 3,5-CBA degradation is the cometabolic degradation by consortia 2m.c [60]. 3,5-CBA was cometabolically converted to the 3,5-dichlorocatechol as a final intermediate by enzyme 2-halobenzoate-1,2-dioxygenase with presence of 2-CBA and 2,5-CBA. Presence of 3,5-

Cl

OH

acid

H2O

COOH

HCl

*IV*

*III*

CoA-SH

H2O

OH

SO CoA

4-hydroxybenzoate-CoA

SO CoA

are degraded cometabolically.

10 Applied Bioremediation - Active and Passive Approaches

ously described in section 2.2.2.

COOH

Cl

2,4-dichlorobenzoic

acid

TCA

Cl

ATP

AMP + PPi

Strain *Pseudomonas putida* P111 degraded 2,3,5-CBA to the 3,5-dichlorocatechol (Figure 12.) but use different degradation pathway than strain *Brevibacterium* sp., 2,3,5-CBA was degraded by 1,2-dioxygenation followed by dehalogenation [62]. Figure 11.Cometabolic degradation of 2,3,6-trichlorobenzoic acid [1]. Strain *Pseudomonas putida* P111 degraded 2,3,5-CBA to the 3,5-dichlorocatechol (Figure 12.) but use different degradation pathway

than strain *Brevibacterium* sp., 2,3,5-CBA was degraded by 1,2-dioxygenation followed by dehalogenation [62].

OH

2,3,5-trichlorophenol

OH

OH

Figure 12.Degradation of 2,3,5-trichlorobenzoic acid by strain *Pseudomonas putida* P111 by 1,2-dioxygenase reaction [62]. **Figure 12.** Degradation of 2,3,5-trichlorobenzoic acid by strain *Pseudomonas putida* P111 by 1,2-dioxygenase reac‐ tion [62].

**2.2.5. Degradation of main chlorobenzoic acids degradation intermediates** 

In CBAs degradation are formed catechol, 3-chlorocatechol, 4-chlorocatechol, 4-hydroxybenzoic acid, gentisic acid, protocatechuic

Catechols can be degraded via *ortho*- or *meta*- cleavage pathway or possibly by modified *ortho-* cleavage pathway, which leads to the 3-oxoadipoic acid pathway. 3-oxoadipoic acid pathway is used for 4-hydroxybenzoic acid and protocatechuc acid degradation

In *ortho*- cleavage pathway is catechol or chlorocatechol degraded by catechol-1,2-dioxygenase or chlorocatechol-1,2-dioxygenase (Figure 13.). The first step of this pathway is dioxygenase reaction leading to the cleavage of the bond between the first and second position of the benzene ring. This reaction produces *cis,cis*-muconic acid or chloro-*cis,cis*-muconic acid with chlorine atom in a

#### *2.2.5. Degradation of main chlorobenzoic acids degradation intermediates*

In CBAs degradation are formed catechol, 3-chlorocatechol, 4-chlorocatechol, 4-hydroxyben‐ zoic acid, gentisic acid, protocatechuic acid, chloroprotocatechuic acid and more chlorinated catechols.

In *ortho*- cleavage pathway chloromuconate cycloisomerase catalyzes the conversion of chlorinated muconic acid to the *cis*- or *trans*-dienelactone in dependence of chlorine atom

Further enzyme of *ortho-* cleavage pathway is the dienelactone hydrolase. This enzyme is capable of conversion of *cis*- or *trans*-dienelactone to the maleylacetate. Maleylacetate is by maleylacetate reductase transformed into 3-oxoadipoic acid, which is further eliminated by 3-

Majority of known strains, use for the catabolism of catechols or chlorocatechols a modified *ortho-* cleavage pathway [64]. In this pathway is chloro-*cis,cis*-muconate transformed by chloromuconate cycloisomerase to the protoanemonine (Figure 13.). Protoanemonine is a dead-end product of this pathway. Protoanemonine has also antimicrobial properties thus formation of protoanemonine from chlorinated catechols in modified *ortho*- cleavage pathway

Catechol or chlorocatechols can be also degraded by the enzyme katechol-2,3-dioxygenase or chlorocatechol-2,3-dioxygenase in *meta-* cleavage pathway which is initiated by this dioxyge‐ nation (Figure 14.). The result of dioxygenase reaction is the ring cleavage between second and

From catechol and 3-chlorocatechol is in this step 3-hydroxy-*cis,cis*-muconic acid formed. This acid is further degraded to the pyruvate and acetaldehyde [65]. The risk of this pathway is the possibility of formation of a reactive acylchloride from 3-chlorocatechol. Acylchloride irrever‐

OH

2-oxopent-4-enoate acid 2-oxohexa-4-enedienoate

catechol

COOH COOH

O

COOH

COOH

COOH

OH

CH2

O

O

chloroacetaldehyde

O

COOH

CH3

O

Bioremediation of Chlorobenzoic Acids http://dx.doi.org/10.5772/56394 13

COOH

Cl

TCA

acetaldehyde

chloroacetic

acid

O

3CH

COOH

Cl

3CH

4-hydroxy-

2-oxopentanoic acid

pyruvate

O

Cl

5-chloro-2-hydroxy-

OH

2,4-dienoic acid 2-oxopentanoic acid

OH

Cl

**Figure 14.** *Meta*- cleavage pathway of catechol or chlorotachols [9]. C23O - catechol-2,3-dioxygenase; CC23O - chlor‐

The 4-chlorocatechol is by the 2,3-dioxygenase reaction converted to the 5-chloro-2-hydroxy‐ muconic semialdehyde [16]. This semialdehyde is in several steps transformed into the

position in muconic acid. In this step is the molecule also dehalogenated [9].

oxoadipoic acid degradation pathway (will be described later).

leads to the poor survival of degrading microorganisms in the soil.

third position of the benzene ring.

OH

OH

4-chlorocatechol 3-chlorocatechol

Cl

OH

CC23O

C23O

CC23O C23O

OH

sibly inactivate catechol-2,3-dioxygenase [66, 67].

Cl OH

COOH

COOH COOH

COOH COOH

> O C23O

C23O

OH

2-hydroxy-

*cis,cis*-muconic

OH

Cl

5-chloro-2-hydroxymuconic acid

5-chloro-2-hydroxy- 5-chloro-2-hydroxy penta-

OH

COOH COCl

COOH CHO

acylchloride

OH

Cl

muconic semialdehyde

ocatechol-2,3-dioxygenase; TCA – tricarboxylic acid cycle.

OH

Catechols can be degraded via *ortho*- or *meta*- cleavage pathway or possibly by modified *ortho*cleavage pathway, which leads to the 3-oxoadipoic acid pathway. 3-oxoadipoic acid pathway is used for 4-hydroxybenzoic acid and protocatechuc acid degradation and leads to the tricarboxylic acid cycle.

In *ortho*- cleavage pathway is catechol or chlorocatechol degraded by catechol-1,2-dioxygenase or chlorocatechol-1,2-dioxygenase (Figure 13.). The first step of this pathway is dioxygenase reaction leading to the cleavage of the bond between the first and second position of the benzene ring. This reaction produces *cis,cis*-muconic acid or chloro-*cis,cis*-muconic acid with chlorine atom in a different position [63].

Figure 13.*Ortho*- and modified *ortho*- cleavage pathway of catechol or chlorotachols adapted from [9]. C12O – catechol-1,2-dioxygenase; CC12O – chlorocatechol-1,2-dioxygenase; MCI – muconate cycloisomerase; CMCI – chloromuconate cycloisomerase; DLH – dienelaktone hydrolase; MAR maleylacetate reductase. **Figure 13.** *Ortho*- and modified *ortho*- cleavage pathway of catechol or chlorotachols adapted from [9]. C12O – cate‐ chol-1,2-dioxygenase; CC12O – chlorocatechol-1,2-dioxygenase; MCI – muconate cycloisomerase; CMCI – chloromuc‐ onate cycloisomerase; DLH – dienelaktone hydrolase; MAR - maleylacetate reductase.

The second enzyme of this pathway is muconate cycloisomerase or chloromuconate cycloisomerase. This is a key enzyme in the degradation of chlorinated substances. The specificity of this isomerase determines whether degradation will be by *ortho*- cleavage pathway or by a modified *ortho-* cleavage pathway. The second enzyme of this pathway is muconate cycloisomerase or chloromuconate cycloiso‐ merase. This is a key enzyme in the degradation of chlorinated substances. The specificity of this isomerase determines whether degradation will be by *ortho*- cleavage pathway or by a modified *ortho-* cleavage pathway.

eliminated by 3-oxoadipoic acid degradation pathway (will be described later).

chlorocatechol. Acylchloride irreversibly inactivate catechol-2,3-dioxygenase [66, 67].

microorganisms in the soil.

between second and third position of the benzene ring.

In *ortho*- cleavage pathway chloromuconate cycloisomerase catalyzes the conversion of chlorinated muconic acid to the *cis*- or *trans*-

Further enzyme of *ortho-* cleavage pathway is the dienelactone hydrolase. This enzyme is capable of conversion of *cis*- or *trans*dienelactone to the maleylacetate. Maleylacetate is by maleylacetate reductase transformed into 3-oxoadipoic acid, which is further

Majority of known strains, use for the catabolism of catechols or chlorocatechols a modified *ortho-* cleavage pathway [64]. In this pathway is chloro-*cis,cis*-muconate transformed by chloromuconate cycloisomerase to the protoanemonine (Figure 13.). Protoanemonine is a dead-end product of this pathway. Protoanemonine has also antimicrobial properties thus formation of protoanemonine from chlorinated catechols in modified *ortho*- cleavage pathway leads to the poor survival of degrading

Catechol or chlorocatechols can be also degraded by the enzyme katechol-2,3-dioxygenase or chlorocatechol-2,3-dioxygenase in *meta-* cleavage pathway which is initiated by this dioxygenation (Figure 14.). The result of dioxygenase reaction is the ring cleavage

From catechol and 3-chlorocatechol is in this step 3-hydroxy-*cis,cis*-muconic acid formed. This acid is further degraded to the pyruvate and acetaldehyde [65]. The risk of this pathway is the possibility of formation of a reactive acylchloride from 3-

dienelactone in dependence of chlorine atom position in muconic acid. In this step is the molecule also dehalogenated [9].

In *ortho*- cleavage pathway chloromuconate cycloisomerase catalyzes the conversion of chlorinated muconic acid to the *cis*- or *trans*-dienelactone in dependence of chlorine atom position in muconic acid. In this step is the molecule also dehalogenated [9].

*2.2.5. Degradation of main chlorobenzoic acids degradation intermediates*

catechols.

tricarboxylic acid cycle.

maleylacetate reductase.

OH

3-chlorocatechol

OH

catechol

Cl

4-chlorocatechol

OH

OH

C12O

OH

C12O CC12O

OH

Cl

HOOC

2-chloro- *cis,cis-*muconic

acid

HOOC

*cis,cis-*muconic acid

HOOC Cl

modified *ortho-* cleavage pathway.

3-chloro-*cis,cis-*muconic

Cl

C12O CC12O

microorganisms in the soil.

pathway or by a modified *ortho-* cleavage pathway.

between second and third position of the benzene ring.

eliminated by 3-oxoadipoic acid degradation pathway (will be described later).

chlorocatechol. Acylchloride irreversibly inactivate catechol-2,3-dioxygenase [66, 67].

chlorine atom in a different position [63].

12 Applied Bioremediation - Active and Passive Approaches

COOH

MCI CMCI O

Cl

O

O

muconolactone

acid 4-chloromuconolactone

O

O

O

5-chloromuconolactone

COOH

O

onate cycloisomerase; DLH – dienelaktone hydrolase; MAR - maleylacetate reductase.

Cl

O

COOH

COOH

Cl

O O

CH2

protoanemonine

COOH

2-chloromuconolactone

COOH

MCI

CMCI

COOH

MCI CMCI

In CBAs degradation are formed catechol, 3-chlorocatechol, 4-chlorocatechol, 4-hydroxyben‐ zoic acid, gentisic acid, protocatechuic acid, chloroprotocatechuic acid and more chlorinated

Catechols can be degraded via *ortho*- or *meta*- cleavage pathway or possibly by modified *ortho*cleavage pathway, which leads to the 3-oxoadipoic acid pathway. 3-oxoadipoic acid pathway is used for 4-hydroxybenzoic acid and protocatechuc acid degradation and leads to the

In *ortho*- cleavage pathway is catechol or chlorocatechol degraded by catechol-1,2-dioxygenase or chlorocatechol-1,2-dioxygenase (Figure 13.). The first step of this pathway is dioxygenase reaction leading to the cleavage of the bond between the first and second position of the benzene ring. This reaction produces *cis,cis*-muconic acid or chloro-*cis,cis*-muconic acid with

O

O O

**Figure 13.** *Ortho*- and modified *ortho*- cleavage pathway of catechol or chlorotachols adapted from [9]. C12O – cate‐ chol-1,2-dioxygenase; CC12O – chlorocatechol-1,2-dioxygenase; MCI – muconate cycloisomerase; CMCI – chloromuc‐

The second enzyme of this pathway is muconate cycloisomerase or chloromuconate cycloiso‐ merase. This is a key enzyme in the degradation of chlorinated substances. The specificity of this isomerase determines whether degradation will be by *ortho*- cleavage pathway or by a

CH2

protoanemonine

O

*cis*-dienelactone

O

HOOC

COOH

*trans*-dienelactone

DLH

DLH

DLH

O

HOOC

COOH

MAR

maleylacetate 3-oxoadipate

O

HOOC

COOH

O

Figure 13.*Ortho*- and modified *ortho*- cleavage pathway of catechol or chlorotachols adapted from [9]. C12O – catechol-1,2-dioxygenase; CC12O – chlorocatechol-1,2-dioxygenase; MCI – muconate cycloisomerase; CMCI – chloromuconate cycloisomerase; DLH – dienelaktone hydrolase; MAR -

The second enzyme of this pathway is muconate cycloisomerase or chloromuconate cycloisomerase. This is a key enzyme in the degradation of chlorinated substances. The specificity of this isomerase determines whether degradation will be by *ortho*- cleavage

In *ortho*- cleavage pathway chloromuconate cycloisomerase catalyzes the conversion of chlorinated muconic acid to the *cis*- or *trans*-

Further enzyme of *ortho-* cleavage pathway is the dienelactone hydrolase. This enzyme is capable of conversion of *cis*- or *trans*dienelactone to the maleylacetate. Maleylacetate is by maleylacetate reductase transformed into 3-oxoadipoic acid, which is further

Majority of known strains, use for the catabolism of catechols or chlorocatechols a modified *ortho-* cleavage pathway [64]. In this pathway is chloro-*cis,cis*-muconate transformed by chloromuconate cycloisomerase to the protoanemonine (Figure 13.). Protoanemonine is a dead-end product of this pathway. Protoanemonine has also antimicrobial properties thus formation of protoanemonine from chlorinated catechols in modified *ortho*- cleavage pathway leads to the poor survival of degrading

Catechol or chlorocatechols can be also degraded by the enzyme katechol-2,3-dioxygenase or chlorocatechol-2,3-dioxygenase in *meta-* cleavage pathway which is initiated by this dioxygenation (Figure 14.). The result of dioxygenase reaction is the ring cleavage

From catechol and 3-chlorocatechol is in this step 3-hydroxy-*cis,cis*-muconic acid formed. This acid is further degraded to the pyruvate and acetaldehyde [65]. The risk of this pathway is the possibility of formation of a reactive acylchloride from 3-

dienelactone in dependence of chlorine atom position in muconic acid. In this step is the molecule also dehalogenated [9].

Further enzyme of *ortho-* cleavage pathway is the dienelactone hydrolase. This enzyme is capable of conversion of *cis*- or *trans*-dienelactone to the maleylacetate. Maleylacetate is by maleylacetate reductase transformed into 3-oxoadipoic acid, which is further eliminated by 3 oxoadipoic acid degradation pathway (will be described later).

Majority of known strains, use for the catabolism of catechols or chlorocatechols a modified *ortho-* cleavage pathway [64]. In this pathway is chloro-*cis,cis*-muconate transformed by chloromuconate cycloisomerase to the protoanemonine (Figure 13.). Protoanemonine is a dead-end product of this pathway. Protoanemonine has also antimicrobial properties thus formation of protoanemonine from chlorinated catechols in modified *ortho*- cleavage pathway leads to the poor survival of degrading microorganisms in the soil.

Catechol or chlorocatechols can be also degraded by the enzyme katechol-2,3-dioxygenase or chlorocatechol-2,3-dioxygenase in *meta-* cleavage pathway which is initiated by this dioxyge‐ nation (Figure 14.). The result of dioxygenase reaction is the ring cleavage between second and third position of the benzene ring.

From catechol and 3-chlorocatechol is in this step 3-hydroxy-*cis,cis*-muconic acid formed. This acid is further degraded to the pyruvate and acetaldehyde [65]. The risk of this pathway is the possibility of formation of a reactive acylchloride from 3-chlorocatechol. Acylchloride irrever‐ sibly inactivate catechol-2,3-dioxygenase [66, 67].

**Figure 14.** *Meta*- cleavage pathway of catechol or chlorotachols [9]. C23O - catechol-2,3-dioxygenase; CC23O - chlor‐ ocatechol-2,3-dioxygenase; TCA – tricarboxylic acid cycle.

The 4-chlorocatechol is by the 2,3-dioxygenase reaction converted to the 5-chloro-2-hydroxy‐ muconic semialdehyde [16]. This semialdehyde is in several steps transformed into the CC23O C23O

OH

OH

OH

4-chlorocatechol

3-chlorocatechol

OH

CC23O

C23O

pyruvate and chloroacetic acid. Pyruvate is further degraded in the tricarboxylic acid cycle. In case of chloroacetic acid is expected further degradation due to the fact that during the course of degradation of 4-CBA via 4-chlorocatechol by strain *Pseudomonas cepacia* P166 only a temporary accumulation of chloroacetic acid was registered [68]. Figure 14.*Meta*- cleavage pathway of catechol or chlorotachols [9]. C23O - catechol-2,3-dioxygenase; CC23O - chlorocatechol-2,3-dioxygenase; TCA – tricarboxylic acid cycle. The 4-chlorocatechol is by the 2,3-dioxygenase reaction converted to the 5-chloro-2-hydroxymuconic semialdehyde [16]. This

COOH

COOH COOH

COOH COOH

> O C23O

C23O

OH

2-hydroxy-

*cis,cis*-muconic

OH

Cl

5-chloro-2-hydroxymuconic acid

5-chloro-2-hydroxy- 5-chloro-2-hydroxy penta-

OH

OH

2-oxopent-4-enoate acid 2-oxohexa-4-enedienoate

catechol

COOH COOH

O

COOH

COOH

COOH

HO

CH2

O

O

chloroacetaldehyde

O

COOH

CH3

O

COOH

Cl

TCA

acetaldehyde

chloroacetic

acid

O

H3C

COOH

ates [9].

as negative.

tive hydrolase transformed into the malate or pyruvate, the tricarboxylic acid cycle intermedi‐

**Figure 16.** Gentisic acid degradation pathway [9]. *I* – gentisate-1,2-dioxygenase; *II* – maleyl pyruvate-*cis,trans*–isomer‐ ase; *III* – maleyl pyruvate-dehydrogenase; *IV* – fumaryl pyruvate-dehydrogenase; TCA – tricarboxylic acid cycle

COOH

O

O

*II*

HOOC

fumaryl pyruvate

COOH

COOH

maleyl pyruvate

OH

H2O

OH

3CH

pyruvate

*III*

H+ H2O

*IV*

COOH

COOH

COOH

malate

fumarate

COOH

TCA

Bioremediation of Chlorobenzoic Acids http://dx.doi.org/10.5772/56394 15

O

H+

HOOC

Due to the complexity of the described CBAs degradation pathways it can be expected that the CBAs degradation pathways will affect each other. This interaction can be positive as well

CBAs itself can affect microbial degradation of xenobiotics which are CBAs degradation products. For example, microbial degradation of PCBs can be by high CBAs concentration

The influence of monochlorinated CBAs on the degradation of low chlorinated PCBs congeners by five different bacterial strains is already documented [15]. It was found that 3-CBA is the most potent inhibitor. The same result was obtained with monochlorinated PCBs degradation by strain *Pseudomonas stutzeri* [70], but in the case of microbial degradation of dichlorinated and trichlorinated PCBs was the inhibition effect of CBAs less significant. In the case of tests with *Pseudomonas testosteroni* B-356 and impact of monochlorinated CBAs on low chlorinated PCBs degradation, it was again found that the 3-CBA has the highest inhibition effect followed

These findings can be explained by the formation of chlorocatechols in 3-CBA degradation with a higher probability than from other monochlorinated CBAs. Degradation of chloroca‐ techols in *meta*- cleavage pathway can lead to the reactive acylchloride formation. Acylchloride can inhibited 2,3-dihydroxybifenyl-1,2-dioxygenase from the upper cometabolic PCBs

*2.2.6. Inhibition and activation of chlorobenzoic acids degradation*

COOH

gentisic acid

OH

OH

O2 H+

*I*

by 4-CBA and the least inhibition showed 2-CBA [16].

slowed down or even stopped [15, 44].

Cl

H3C

4-hydroxy-

2-oxopentanoic acid

pyruvate

O

Cl

5-chloro-2-hydroxy-

HO

2,4-dienoic acid 2-oxopentanoic acid

OH

Cl

fourth position. This way formed 3-karboxy-*cis*,*cis*-muconic acid is than transformed into the 3-oxoadipoic acid [51, 69].

Cl OH

COOH COCl

COOH CHO

acylchloride

OH

muconic semialdehyde

OH

In CBAs degradation next to catechol and chlorocatechols can be 4-hydroxybenzoic acid formed. 4-hydroxybenzoic acid is product of 4-CBA degradation (section 2.2.3). 4-hydroxy‐ benzoic acid is further degraded in the 3-oxoadipoic acid degradation pathway (Figure 15.) [16]. The first step of this pathway is oxidation catalyzed by 4-hydroxybenzoate-3-monooxy‐ genase with the formation of protocatechuic acid (3,4-dihydroxybenzoic acid). Benzene ring of protocatechuic acid is in next step cleaved between third and fourth position. This way formed 3-karboxy-*cis*,*cis*-muconic acid is than transformed into the 3-oxoadipoic acid [51, 69]. semialdehyde is in several steps transformed into the pyruvate and chloroacetic acid. Pyruvate is further degraded in the tricarboxylic acid cycle. In case of chloroacetic acid is expected further degradation due to the fact that during the course of degradation of 4-CBA via 4-chlorocatechol by strain *Pseudomonas cepacia* P166 only a temporary accumulation of chloroacetic acid was registered [68]. In CBAs degradation next to catechol and chlorocatechols can be 4-hydroxybenzoic acid formed. 4-hydroxybenzoic acid is product of 4-CBA degradation (section 2.2.3). 4-hydroxybenzoic acid is further degraded in the 3-oxoadipoic acid degradation pathway (Figure 15.) [16]. The first step of this pathway is oxidation catalyzed by 4-hydroxybenzoate-3-monooxygenase with the formation of protocatechuic acid (3,4-dihydroxybenzoic acid). Benzene ring of protocatechuic acid is in next step cleaved between third and

Figure 15.Degradation pathway of 3-oxoadipoic acid [9, 51, 69]. *I* – 4-hydroxybenzoate-3-monooxygenase; *II* – protokatechute-3,4-dioxygenase; *III* – 3-carboxymuconate-cykloisomerase; *IV* – 4-carboxymuconolaktone-dekarboxylase; *V* – 3-oxoadipate-enol-laktone hydrolase; *VI* - 3 oxoadipate:sukcinyl-CoA transferase; *VII* - 3-oxoadipate-CoA thiolase; TCA – tricarboxylic acid cycle. **Figure 15.** Degradation pathway of 3-oxoadipoic acid [9, 51, 69]. *I* – 4-hydroxybenzoate-3-monooxygenase; *II* – proto‐ katechute-3,4-dioxygenase; *III* – 3-carboxymuconate-cykloisomerase; *IV* – 4-carboxymuconolaktone-dekarboxylase; *V* – 3-oxoadipate-enol-laktone hydrolase; *VI* - 3-oxoadipate:sukcinyl-CoA transferase; *VII* - 3-oxoadipate-CoA thiolase; TCA – tricarboxylic acid cycle.

3-oxoadipoic acid, which is also formed in the *ortho*- cleavage pathway from chlorinated catechols is then in two steps catalyzed by 3-oxoadipate:succinyl-CoA transferase and 3-oxoadipoate-CoA thiolase converted into the intermediates of the trikarboxylic acid cycle [9, 51, 69]. In 3CBA degradation pathway initiated by monoxygenase reaction is gentisic acid formed (section 2.2.2). Gentisic acid (2,5 dihydroxybenzoic acid) is, as well as protocatechuic acid, degraded in the 3-oxoadipoic acid degradation pathway into the 3-oxoadipoic acid, which is also formed in the *ortho*- cleavage pathway from chlorinated catechols is then in two steps catalyzed by 3-oxoadipate:succinyl-CoA transferase and 3 oxoadipoate-CoA thiolase converted into the intermediates of the trikarboxylic acid cycle [9, 51, 69].

intermediates of the tricarboxylic acid cycle (Figure 16.). In 3-CBA degradation pathway initiated by monoxygenase reaction is gentisic acid formed (section 2.2.2). Gentisic acid (2,5-dihydroxybenzoic acid) is, as well as protocatechuic acid, degraded in the 3-oxoadipoic acid degradation pathway into the intermediates of the tricar‐ boxylic acid cycle (Figure 16.).

The first step in gentisic acid degradation is the formation of maleylpyruvate by dioxygenase reaction catalyzed by gentisate-1,2-dioxygenase. Maleylpyruvate can be by isomerization transformed into the fumarylpyruvate. Maleylpyruvate and fumarylpyruvate are by respec‐

**Figure 16.** Gentisic acid degradation pathway [9]. *I* – gentisate-1,2-dioxygenase; *II* – maleyl pyruvate-*cis,trans*–isomer‐ ase; *III* – maleyl pyruvate-dehydrogenase; *IV* – fumaryl pyruvate-dehydrogenase; TCA – tricarboxylic acid cycle

tive hydrolase transformed into the malate or pyruvate, the tricarboxylic acid cycle intermedi‐ ates [9].

#### *2.2.6. Inhibition and activation of chlorobenzoic acids degradation*

pyruvate and chloroacetic acid. Pyruvate is further degraded in the tricarboxylic acid cycle. In case of chloroacetic acid is expected further degradation due to the fact that during the course of degradation of 4-CBA via 4-chlorocatechol by strain *Pseudomonas cepacia* P166 only a

Cl

5-chloro-2-hydroxymuconic acid

5-chloro-2-hydroxy- 5-chloro-2-hydroxy penta-

COOH

COOH COOH

COOH COOH

> O C23O

C23O

OH

2-hydroxy-

*cis,cis*-muconic

OH

OH

OH

2-oxopent-4-enoate acid 2-oxohexa-4-enedienoate

catechol

COOH COOH

O

COOH

COOH

COOH

HO

CH2

O

O

chloroacetaldehyde

O

COOH

CH3

O

COOH

Cl

TCA

acetaldehyde

chloroacetic

acid

O

H3C

COOH

Cl

H3C

4-hydroxy-

2-oxopentanoic acid

pyruvate

O

Cl

5-chloro-2-hydroxy-

HO

2,4-dienoic acid 2-oxopentanoic acid

OH

Cl

Figure 14.*Meta*- cleavage pathway of catechol or chlorotachols [9]. C23O - catechol-2,3-dioxygenase; CC23O - chlorocatechol-2,3-dioxygenase; TCA

The 4-chlorocatechol is by the 2,3-dioxygenase reaction converted to the 5-chloro-2-hydroxymuconic semialdehyde [16]. This semialdehyde is in several steps transformed into the pyruvate and chloroacetic acid. Pyruvate is further degraded in the tricarboxylic acid cycle. In case of chloroacetic acid is expected further degradation due to the fact that during the course of degradation of 4-CBA via 4-chlorocatechol by strain *Pseudomonas cepacia* P166 only a temporary accumulation of chloroacetic acid

In CBAs degradation next to catechol and chlorocatechols can be 4-hydroxybenzoic acid formed. 4-hydroxybenzoic acid is product of 4-CBA degradation (section 2.2.3). 4-hydroxybenzoic acid is further degraded in the 3-oxoadipoic acid degradation pathway (Figure 15.) [16]. The first step of this pathway is oxidation catalyzed by 4-hydroxybenzoate-3-monooxygenase with the formation of protocatechuic acid (3,4-dihydroxybenzoic acid). Benzene ring of protocatechuic acid is in next step cleaved between third and

COOH

O

3-carboxymuconolactone

*IV*

*V*

O

3-oxoadipateenol-laktone

COOH

COOH COOH

3-oxoadipic acid

O

fourth position. This way formed 3-karboxy-*cis*,*cis*-muconic acid is than transformed into the 3-oxoadipoic acid [51, 69].

HOOC

O

Cl OH

COOH COCl

COOH CHO

acylchloride

OH

Cl

muconic semialdehyde

OH

In CBAs degradation next to catechol and chlorocatechols can be 4-hydroxybenzoic acid formed. 4-hydroxybenzoic acid is product of 4-CBA degradation (section 2.2.3). 4-hydroxy‐ benzoic acid is further degraded in the 3-oxoadipoic acid degradation pathway (Figure 15.) [16]. The first step of this pathway is oxidation catalyzed by 4-hydroxybenzoate-3-monooxy‐ genase with the formation of protocatechuic acid (3,4-dihydroxybenzoic acid). Benzene ring of protocatechuic acid is in next step cleaved between third and fourth position. This way formed 3-karboxy-*cis*,*cis*-muconic acid is than transformed into the 3-oxoadipoic acid [51, 69].

> OH COOH COOH

> > HOOC

O

*VII VI*

HSCoA HSCoA

3-oxoadipate-CoA

*I II III*

COOH

COSCoA

acid

3-carboxy-*cis,cis*-muconic

oxoadipate:sukcinyl-CoA transferase; *VII* - 3-oxoadipate-CoA thiolase; TCA – tricarboxylic acid cycle.

3-oxoadipoic acid, which is also formed in the *ortho*- cleavage pathway from chlorinated catechols is then in two steps catalyzed by 3-oxoadipate:succinyl-CoA transferase and 3 oxoadipoate-CoA thiolase converted into the intermediates of the trikarboxylic acid cycle [9,

In 3-CBA degradation pathway initiated by monoxygenase reaction is gentisic acid formed (section 2.2.2). Gentisic acid (2,5-dihydroxybenzoic acid) is, as well as protocatechuic acid, degraded in the 3-oxoadipoic acid degradation pathway into the intermediates of the tricar‐

The first step in gentisic acid degradation is the formation of maleylpyruvate by dioxygenase reaction catalyzed by gentisate-1,2-dioxygenase. Maleylpyruvate can be by isomerization transformed into the fumarylpyruvate. Maleylpyruvate and fumarylpyruvate are by respec‐

**Figure 15.** Degradation pathway of 3-oxoadipoic acid [9, 51, 69]. *I* – 4-hydroxybenzoate-3-monooxygenase; *II* – proto‐ katechute-3,4-dioxygenase; *III* – 3-carboxymuconate-cykloisomerase; *IV* – 4-carboxymuconolaktone-dekarboxylase; *V* – 3-oxoadipate-enol-laktone hydrolase; *VI* - 3-oxoadipate:sukcinyl-CoA transferase; *VII* - 3-oxoadipate-CoA thiolase;

O

intermediates of the tricarboxylic acid cycle (Figure 16.).

temporary accumulation of chloroacetic acid was registered [68].

COOH

OH

H3C COSCoA

protocatechuic 4-hydroxybenzoic

acid acid

acetyl-CoA

CoASOC

HOOC

succinyl-CoA

OH

OH

4-chlorocatechol

3-chlorocatechol

Cl

OH

CC23O

C23O

CC23O C23O

14 Applied Bioremediation - Active and Passive Approaches

OH

– tricarboxylic acid cycle.

was registered [68].

COOH

OH

TCA

cycle [9, 51, 69].

boxylic acid cycle (Figure 16.).

TCA – tricarboxylic acid cycle.

51, 69].

Due to the complexity of the described CBAs degradation pathways it can be expected that the CBAs degradation pathways will affect each other. This interaction can be positive as well as negative.

Figure 15.Degradation pathway of 3-oxoadipoic acid [9, 51, 69]. *I* – 4-hydroxybenzoate-3-monooxygenase; *II* – protokatechute-3,4-dioxygenase; *III* – 3-carboxymuconate-cykloisomerase; *IV* – 4-carboxymuconolaktone-dekarboxylase; *V* – 3-oxoadipate-enol-laktone hydrolase; *VI* - 3- CBAs itself can affect microbial degradation of xenobiotics which are CBAs degradation products. For example, microbial degradation of PCBs can be by high CBAs concentration slowed down or even stopped [15, 44].

3-oxoadipoic acid, which is also formed in the *ortho*- cleavage pathway from chlorinated catechols is then in two steps catalyzed by 3-oxoadipate:succinyl-CoA transferase and 3-oxoadipoate-CoA thiolase converted into the intermediates of the trikarboxylic acid In 3CBA degradation pathway initiated by monoxygenase reaction is gentisic acid formed (section 2.2.2). Gentisic acid (2,5 dihydroxybenzoic acid) is, as well as protocatechuic acid, degraded in the 3-oxoadipoic acid degradation pathway into the The influence of monochlorinated CBAs on the degradation of low chlorinated PCBs congeners by five different bacterial strains is already documented [15]. It was found that 3-CBA is the most potent inhibitor. The same result was obtained with monochlorinated PCBs degradation by strain *Pseudomonas stutzeri* [70], but in the case of microbial degradation of dichlorinated and trichlorinated PCBs was the inhibition effect of CBAs less significant. In the case of tests with *Pseudomonas testosteroni* B-356 and impact of monochlorinated CBAs on low chlorinated PCBs degradation, it was again found that the 3-CBA has the highest inhibition effect followed by 4-CBA and the least inhibition showed 2-CBA [16].

> These findings can be explained by the formation of chlorocatechols in 3-CBA degradation with a higher probability than from other monochlorinated CBAs. Degradation of chloroca‐ techols in *meta*- cleavage pathway can lead to the reactive acylchloride formation. Acylchloride can inhibited 2,3-dihydroxybifenyl-1,2-dioxygenase from the upper cometabolic PCBs

degradation pathway (Figure 1.). How was proved by accumulation of hydroxylated biphen‐ yls during PCBs degradation by strain *Pseudomonas testosteroni* B-356 [16].

Acylchloride can be also irreversibly bound on catechol or chlorocatechol-2,3-dioxygenase, the first enzyme in the *meta*- cleavage pathway. Its inactivation leads to catechols or chlorocate‐ chols accumulation and therefore to the inhibition of CBAs degradation [71].

Another potentially dangerous intermediate is protoanemonine, substance with antibiotic properties. Protoanemonine is formed in the modified *ortho*- cleavage pathway from muconic or chloromuconic acid. The main effect of protoanemonine is growth inhibition of degrading microorganisms[72].

The presence of a mixture of CBAs can also cause inhibition or activation of CBA degradation. Strain *Burkholderia cepacia* JHR22 can degrade 2-CBA, 3-CBA, 4-CBA and 3,5-CBA [44] when present individually. This strain loses 2-CBA degradation ability when 2-CBA is present in mixture with 2,3-CBA or 3,4-CBA. On the other hand addition of 2,5-CBA or 2,6-CBA had no effect on 2-CBA degradation. When was 2-CBA added with 2,4-CBA strain *Burkholderia cepacia* JHR22 in addition to the 2-CBA degradation can cometabolically degrade 2,4-CBA.

Catechols or chlorocatechols are also potential inhibitors of bacterial CBAs degradation. If these intermediates of CBAs degradation are not enough quickly degraded they can be subject to the auto-oxidation or enzymatic polymerization with formation of brown or black pigment [73, 74]. The presence of this pigment inhibits CBAs degradation by affecting the shape of bacterial cells which may consequently lead to their death as in case of strain *Pseudomonas fluorescens* [75].
