Microbial degradation of azo dyes
Since sulphonated azo dyes are apparently only degraded under aerobic conditions in rare exceptional cases, we have developed a strategy for the anaerobic/aerobic treatment of the foreign substances for this group of substances. Here, the ability to anaerobically unspecifically reduce the azo bond, which is widespread among bacteria, is coupled with the specific ability of our isolates to aerobically degrade aminobenzene and aminonaphthalene sulphonates. In the course of these investigations, we were able to achieve microbial mineralisation of sulphonated azo dyes for the first time by a specific anaerobic/aerobic treatment (Haug et al., 1991; Kudlich et al., 1996).
The biological system we developed was subsequently further investigated in terms of process technology in the working group of Prof. Hempel (TU Braunschweig) and formed the basis for a technical system for the treatment of dyeing wastewater in a textile company.
Furthermore, our working group has analysed the molecular basis for the non-specific anaerobic reduction of azo dyes. It was observed that the addition of quinoid redox mediators (especially 2-hydroxy-1,4-naphthoquinone and anthraquinone-2-sulfonate) leads to a significant increase in the anaerobic reduction rates of azo dyes by various bacteria (Kudlich et al., 1997). A model was then developed in which the quinones are reduced enzymatically by the bacteria and the reduced quinones then cleave the azo bonds in the cell-free space in a purely chemical reaction. In further work, the biological reduction of the quinones by the bacteria and the chemical reduction of the azo dyes by the reduced quinones were analysed separately. The applicability of quinoid redox mediators for the decolourisation of azo dyes depends primarily on the redox potential of the quinones and it was possible to define a "redox window" that allows an optimal decolourisation of azo dyes (Rau et al., 2002a).
It could also be shown that low-molecular redox mediators not only enable the reduction of highly polar sulphonated azo dyes but also the reduction of pharmacologically relevant polymeric azo dyes (Rau et al., 2002b). Furthermore, it could be shown that the intracellular reduction of the redox mediator Lawson (2-hydroxy-1,4-naphthoquinone) in E. coli is catalysed by the "oxygen-insensitive" nitroreductases NfsA and NfsB (Rau & Stolz, 2003).
Using the example of the naphthalene sulfonic acid-degrading strain Sphingomonas xenophaga BN6, it was demonstrated that microorganisms also produce active redox mediators as part of their metabolism and we were able to identify amino-1,2-naphthoquinones substituted in the 4-position as extremely effective redox mediators (Keck et al., 1997, 2002).
Furthermore, productive aerobic degradation of a sulphonated azo dye was achieved for the first time in the course of our investigations. After a long-term adaptation phase, the strain Hydrogenophaga intermedia S1, which is involved in the degradation of sulphanilic acid, was adapted to the utilisation of 4-carboxy-4'-sulfoazobenzene (Blümel et al., 1998).
We investigated the molecular basis of the aerobic degradation of azo dyes using the example of the carboxylated azo dye-degrading strains "Pseudomonas" K24 and KF46. The genes of both azoreductases were cloned and the enzymes heterologously expressed in E. coli. These experiments yielded the surprising finding that the two aerobic azoreductases apparently evolved independently of each other (Blümel et al., 2002; Blümel & Stolz, 2003; Bürger & Stolz, 2010).
Microbial degradation of aromatic aminosulfonate
Sulfonated aromatics are produced on a large scale by the chemical industry (e.g. as detergents, dispersants, optical brighteners, ion exchangers and pharmaceuticals).
These compounds are often difficult to degrade in conventional wastewater treatment plants and can therefore be detected in many places in the environment. Within the framework of our investigations, we have elucidated the microbial degradation pathways of substituted aromatic sulphonic acids using the example of the degradation of aminobenzene and aminonaphthalene sulphonates and have purified and characterised various key enzymes involved in the degradation. Furthermore, the genetic basis of these degradation pathways was investigated and new insights into the evolution of the ability to degrade sulfoaromatics were gained. Here, using the example of the degradation of aminonaphthalenesulphonic acids by Sphingomonas xenophaga BN6, it was shown that the naphthalenesulphonates are converted to the corresponding dihydroxynaphthalenes by an initial desulphonation and are then converted to (substituted) salicylates via substituted hydroxychromencarboxylic acids and hydroxybenzalpyruvates.
Detailed enzymatic studies have shown that the development of a productive degradation pathway primarily requires a novel regioselective desulfonating "naphthalene sulfonic acid dioxygenase", while all other required enzymes can be recruited from more widespread degradation pathways (especially for naphthalene). Here, it was shown that a number of enzymes of the naphthalene degradation pathway are capable of turnover of subtitled substrates (Kuhm et al., 1991a,b, 1993a,b; Nörtemann et al., 1994; Stolz, 1999).
The degradation of 4-aminobenzenesulfonic acid (sulfanilic acid) by a bacterial two-species culture from Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2 differs from the degradation pathway of aminonaphthalenesulfonic acids (and almost all other sulfonated aromatics), since in this degradation pathway no initial desulfonation is observed and the xenobiotic sulfonic acid substituent is retained over several enzymatic steps. In this process, 4-sulfocatechol, 3-sulfomuconic acid, "4-sulfomuconolactone" and maleyl acetate were identified as intermediates.
Purification of the enzymes involved proved that 4-sulfocatechol is oxidised to 3-sulfomuconate by modified protocatechuate-3,4-dioxygenases ("type II enzymes") (Hammer et al., 1996). The genes coding for the initial dioxygenases were cloned from both strains and characterised (Contzen & Stolz 2000; Contzen et al., 2001). In further investigations, the genes responsible for the subsequent reactions of 4-sulfocatechol degradation were cloned from the two bacterial strains. Here, the genes for a modified form of the 3-carboxymuconate cycloisomerases ("type II enzymes"), which are also involved in protocatechuate degradation, were identified, the gene products of which can apparently also convert sulphonated muconates. Multiple sequence comparisons showed that the two type II enzymes apparently form a defined subgroup within the 3-carboxymuconate cycloisomerases and exhibit some specific amino acid exchanges.
This proved that a modified 3-ketoadipate pathway is apparently realised in nature for the mineralisation of substituted benzenesulphonates. The genes for the following enzyme in the degradation pathway of the 4-sulphocatechol ("sulphomuconolactone hydrolase") were also identified in both strains. Significant sequence homology with pyrondicarboxylate hydrolases was found for these genes. This suggests a previously undescribed "patchwork assembly" of the 4-sulphocatechol degradation pathway from modified enzymes of the 3-ketoadipate pathway and enzymes of the extradiolic protocatechuate degradation pathway. Furthermore, we and other research groups were able to show that the newly described degradation pathway for 4-sulfocatechol apparently has a wide distribution in the degradation of substituted benzenesulfonates (Contzen et al., 1996, 2001).
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