3-Chloro-, 2,3- and 3,5-dichlorobenzoate co-metabolism in a 2-chlorobenzoate-degrading consortium: role of 3,5-dichlorobenzoate as antagonist of 2-chlorobenzoate degradation

Abstract

A study was made of the metabolic and co-metabolic intermediates of 2- and 3-chlorobenzoate, 2,3- and 3,5-dichlorobenzoate to elucidate the mechanism(s) involved in the negative effects observed on the growth of a chlorobenzoate-degrading microbial consortium in the presence of mixed chlorobenzoates. 2-Chloromuconate accumulated as the end-product in the cultural broths of the microbial consortium during growth on 2-chlorobenzoate; the same 2-chloromuconate was identified in the reaction mixtures of resting cells pregrown on 2-chlorobenzoate and exposed to 3-chloro- and 2,3-dichlorobenzoate, while in similar experiments 1,2-dihydroxy-3,5-dichloro-cyclohexa-3,5-dienoate was detected as dead-end product of 3,5-dichlorobenzoate co-metabolism. These results suggest an initial degradative attack by 2-chlorobenzoate induced dioxygenase(s). The role of 3,5-dichlorobenzoate as an antagonist of 2-chlorobenzoate degradation was also studied: in the presence of mixed 2-chloro- and 3,5-dichlorobenzoate, the 3,5-dichlorobenzoate preferential uptake by the resting cells of the chlorobenzoate-degrading consortium was observed. 2-Chlorobenzoate entered the cells only after the complete removal of the co-substrate. In growing cells experiments, the addition of 1,2-dihydroxy-3,5-dichloro-cyclohexa-3,5-dienoate, the 3,5-dichlorobenzoate co-metabolite, to 2-chlorobenzoate exerted the same antagonistic effect of the parent compound, inhibiting both the microbial growth and the degradative process. These data are discussed, allowing us to attribute the inhibitory effects observed to a substrate/co-substrate competition, though other additional causes may not be totally excluded.

Inhibitory mechanisms by chlorobenzoate mixtures in chlorobenzoate-degrading microorganisms

“…The TLC and 1 H-NMR of the crude organic extracts attributed these intermediates to p-hydroxy-and 3,4-dihydroxybenzoic acids, by comparison with authentic standards. These results show that adaptation to 4CB determined a deep change in the enzyme degradation set of the consortium, which degraded 4CB via protocatechuate and not via the dioxygenation of the chlorinated ring, as previously demonstrated for 2CB and 2,5DCB (Baggi et al, 2005(Baggi et al, , 2008.…”

“…The results obtained show that 3CB, 2,3DCB, 2,5DCB and 2,3,5TCB have the same behaviour as the previously studied 3,5DCB (Baggi et al, 2005), being preferentially taken up by the microbial consortium resting cells. Note that 3,4DCB did not enter the cells, but it hindered the 2CB uptake.…”

“…A microbial consortium, obtained through an enrichment procedure on 2CB, was previously characterised in its growth and metabolic properties (Baggi and Zangrossi, 2001;Baggi et al, 2005Baggi et al, , 2008. Here we have studied some characteristics of the same microbial consortium when it grew on 4CB and 2,5DCB, both utilised as sole carbon source.…”

“…The antagonistic effect of 3,5DCB on microbial consortium growth has been demonstrated to depend on its competition with the growth substrate, as the dihaloaromatic enters the cells preferentially and faster than the said 2CB (Baggi et al, 2005). In order to investigate the effects of different binary CB mixtures on cell uptake mechanisms, experiments were conducted with resting cells of the microbial consortium grown on 2CB, 2,5DCB and 4CB and Alcaligenes denitrificans grown on 4CB.…”

“…The formation of toxic metabolic intermediates such as chlorocatechol(s) and/or their meta-fission product(s) was hypothesized, but this has not always been demonstrated experimentally (Stratford et al, 1996). In other cases, interactions at the level of enzyme induction (Weissenfels et al, 1990, Baggi andZangrossi, 1999), the type of the carbon source utilised for growth (Hernandez et al, 1991) or competitive interferences with uptake systems or key enzymes involved in the first steps of the CB degradative process (Stringfellow and Aitken, 1985;Adriaens and Focht, 1991;Miguez et al, 1995;Corbella et al, 2001;Baggi et al, 2005), were postulated as the most important causes of the inhibitory phenomena.…”