4-Chlorobenzoic acid has a molecular weight of 156.56. At room temperature, it is a white, needle-like crystalline powder with a slightly pungent odor. It is chemically stable and exhibits the typical reactivity of the benzoic acid functional group, allowing for transformations such as esterification, decarboxylation, and substitution. As an important organic synthesis intermediate, it is widely used in the pharmaceutical, pesticide, dye, and fragrance industries for the preparation of antimicrobial agents, herbicides, and chemical additives.

Dehalogenation of 4-Chlorobenzoic Acid by Pseudomonas isolates

Dechlorinase/dehalogenase is the key enzyme in degradation of chlorinated compounds. The carbon-halogen bond is cleaved either by enzymatic dehalogenation, where the biocatalysis of the carbon– halogen bond is achieved by specific enzymes –– the dehalogenases, or by spontaneous chemical dehalogenation of unstable intermediates. In addition, there are enzymes which, because of their relaxed substrate specificity, catalyze the conversion of halogenated analogs of the corresponding unsubstituted substrates or of related compounds, which might lead to ‘fortuitous’ dehalogenation of the substrate analog. Dehalogenases, normally inducible enzymes enable the microorganisms to grow on halogenated aliphatic and aromatic compounds, as their sole carbon and energy source. The bacterial degradation of Chlorobenzoates (CBAs) involve most of the time a rate limiting step of inorganic chloride removal which is species specific and is carried out by a group of enzymes referred to as dehalogenase. The buffered salt solution medium's supplemented with 500ug/ml of 4-chlorobenzoic acid (4-CBA),as a sole source of carbon and energy, was used for studying the growth kinetics. Periodic increase in optical density (540nm)and chloride ion accumulation were measured as function of growth of the organism. The degradation of 4-CBA in the culture medium was monitored spectrophotometrically by measuring chloride ion release in the culture filtrate at 460nm by mercuric thiocynate method described by Bergmann and Sainik (1957) and decrease in 4-chlorobenzoic acid concentration at 260nm.Based on comparative growth response, three pure isolates GVS-4,GVS-18 and GWS-19 and three consortia SW-1,SW-2 and SW-3 were finally selected for further studies.GVS-4,GVS-18 and GWS-19 were identified as Pseudomonas putida and P.aeruginosa respectively at MTCC, IMTECH, Chandigarh. The dechlorinase/dehalogenase activity of all the cultures was estimated in cell free crude extracts by reacting with the different concentration (100 ug/ml -500ug/ml) of 4-chlorobenzoic acid in the reaction mixture.[1]

Enzyme Substrate Kinetics Studies: The dechlorinase/dehalogenase activity of all the cultures was estimated in cell free crude extracts by reacting with the different con- centration (100 ug/ml -500ug/ml)of 4-chlorobenzoic acid in the reaction mixture. Enzyme kinetics of dehalogenase enzyme of Pseudomonas pure strains GVS-4,GVS-18,GWS-19 and consortium SW-2 were studied. A comparatively lower dehalogenase activity at 100ug/ml of 4-chlorobenzoic acid concentration was observed and the activity was found to increase with increase in concentration upto 300ug/ml while constancy was observed on further increase in concentration. Earlier studies also suggested that high substrate concentration in enzyme systems results in low affinities for the substrate as the presence of residual substrate. So, the dioxygenase assay of the Pseudomonas isolates revealed the presence of catechol 1,2 dioxygenase but no catechol 2,3 dioxygenase activity suggesting that 4-CBA is degraded by ortho ring fission. In fact, a large number of workers have reported degradation of 4-chlorobenzoic acid by the formation of catechols as intermediates t that are formed by spontaneous removal of chlorine from chlorocatechols as a result of 1,2 dioxygenase attack as also confirmed.

Biodegradation of 4-chlorobenzoic acid

Chlorinated aromatic compounds are the major chemical pollutants because they enter the environment in large quantities and are toxic and resistant to degradation and bioaccumulate. There are few reports on characterization of bacterial communities capable of degrading chloroaromatic pollutants (Greene et al. 2000; Kubicek et al. 2003; Watanabe et al. 2002). In this paper, we describe the isolation and characterization of bacterial consortium capable of degrading chloroaromatic compounds and the pathway for the degradation of 4-chlorobenzoic acid by Pseudomonas aeruginosa PA01 NC. The metabolites were isolated from culture filtrate of the organism grown on 4-chlorobenzoic acid by extraction with diethyl ether and analysed by thin layer chromatography (TLC) on silica gel G plates using the solvent systems: A) Benzene-acetic acid (85:15, v/v) and B) Benzene-toluene-acetic acid (2:2:1, v/v). The metabolites were visualized under ultraviolet (UV) light (at 254 nm) or by exposure to iodine vapours and also by spraying with 1% FeCl3–K3Fe(CN)6 solution in water. The analysis of the culture extracts of P. aeruginosa PA01 NC grown on 4-chlorobenzoic acid by TLC revealed the presence of a compound, whose Rf value corresponded with that of authentic 4-chlorocatechol. HPLC analysis of isolated metabolite showed the retention time of 3.91 min identical to that of authentic 4-chlorocatechol.[2]

A bacterial consortium capable of degrading chloroaromatic compounds was isolated from pulp and paper mill effluents by selective enrichment on 4-chlorobenzoic acid as sole source of carbon and energy. The four different bacterial isolates obtained from bacterial consortium were identified as Pseudomonas aeruginosa AY792969 (A), P. aeruginosa PA01 NC (B), Pseudomonas sp. ZZ5 DQ113452 (C) and Pseudomonas sp. AY762360 (D) based on their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. These bacterial isolates were found to be versatile in degrading a variety of chloroaromatic compounds including fluoro- and iodobenzoic acids. P. aeruginosa PA01 NC utilized 4-chlorobenzoic acid at 2 g/l as growth substrate. Biodegradation studies have revealed that this organism degraded 4-chlorobenzoic acid through 4-chlorocatechol which was further metabolized by ortho-cleavage pathway and the dechlorination occurred after the ring-cleavage.

References

[1]Banta G, Kahlon RS. Dehalogenation of 4 - Chlorobenzoic Acid by Pseudomonas isolates. Indian J Microbiol. 2007 Jun;47(2):139-43. doi: 10.1007/s12088-007-0027-5. Epub 2007 Jul 8. PMID: 23100656; PMCID: PMC3450107.

[2]Hoskeri RS, Mulla SI, Shouche YS, Ninnekar HZ. Biodegradation of 4-chlorobenzoic acid by Pseudomonas aeruginosa PA01 NC. Biodegradation. 2011 Jun;22(3):509-16. doi: 10.1007/s10532-010-9423-3. Epub 2010 Oct 6. PMID: 20924649.