Azo compounds constitute the largest and the most diverse group of synthetic dyes and are widely used in a number of industries such as textile, food, cosmetics and paper printing. They are generally recalcitrant to biodegradation due to their xenobiotic nature. However microorganisms, being highly versatile, have developed enzyme systems for the decolorization and mineralization of azo dyes under certain environmental conditions. Several genera of Basidomycetes have been shown to mineralize azo dyes. Reductive cleavage of azo bond, leading to the formation of aromatic amines, is the initial reaction during the bacterial metabolism of azo dyes. Anaerobic/anoxic azo dye decolorization by several mixed and pure bacterial cultures have been reported. Under these conditions, this reaction is non-specific with respect to organisms as well as dyes. Various mechanisms, which include enzymatic as well as low molecular weight redox mediators, have been proposed for this non-specific reductive cleavage. Only few aerobic bacterial strains that can utilize azo dyes as growth substrates have been isolated. These organisms generally have a narrow substrate range. Degradation of aromatic amines depends on their chemical structure and the conditions. It is now known that simple aromatic amines can be mineralized under methanogenic conditions. Sulfonated aromatic amines, on the other hand, are resistant and require specialized aerobic microbial consortia for their mineralization. This review is focused on the bacterial decolorization of azo dyes and mineralization of aromatic amines, as well as the application of these processes for the treatment of azo-dye-containing wastewaters.

A variety of environmental inocula were tested for the development of 2-aminobenzenesulfonate (2-ABS, Orthanilic acid) degrading bacterial enrichment. A bacterial consortium (BC), which could utilize 2-ABS as the sole carbon and energy source, could only be developed from the sludge derived from a wastewater treatment unit of a large chemical industry manufacturing nitro and aminoaromatics. BC consisted of two bacterial strains. Based on 16S rDNA sequence analysis, these strains were identified to be belonging to the genus, Acinetobacter and Flavobacterium. The consortium could degrade 1,000 mg l−1 2-ABS within 40 h. Evidence for the extensive mineralization of 2-ABS, during the growth of BC, was derived from U.V-spectral and total organic carbon analysis. BC was highly specific for 2-ABS, as other benzene sulfonates tested in this study, including other ABS isomers, were not utilized as growth substrates. 2-ABS removal pattern in the presence of glucose was significantly influenced by acclimation characteristics of the culture. Consortium adapted to 2-ABS/glucose demonstrated the concomitant removal of both substrates, whereas glucose exerted catabolic repression on 2-ABS removal with glucose adapted culture. Presence of chloramphenicol inhibited 2-ABS degradation by cells, pregrown on succinate, indicating that the 2-ABS degrading enzymes are inducible in nature. Thus the presence of 2-ABS is essential for maintaining the high degradation potential. This enrichment culture can find an application in the treatment of 2-ABS containing wastewaters.

Decolorization of two monoazo dyes, acid orange 6 (AO6) and acid orange 7 (AO7), were studied in sequential fixed-film anaerobic batch reactor (SFABR) with varying dye concentrations and 500mg/L glucose as the co-substrate. More than 90% dye decolorization could be achieved, even at 300mg/L, with both AO6 and AO7 and dye decolorization rates were 168mg/L/d and 176mg/L/d, respectively. COD removals with these two monoazo dyes were significantly different, as 75% and 35% decrease were observed with AO6 and AO7, respectively. UV–visible spectral as well as HPLC analysis of SFABR treated effluent showed the accumulation of 4-aminobenzenesulfonate (4-ABS) from AO6 and AO7. Aminoresorcinol (AR) formed from AO6 decolorization could not be detected at the end of SFABR cycle. This along with high COD removal indicated its further degradation. Formation of pink coloration on exposure to air indicated the presence of 1-amino-2-naphthol (AN) in AO7 fed reactor effluent. Thus both 4-ABS and AN were resistant to further degradation under anaerobic conditions. Presence of nitrate did not decrease the observed decolorization at the end of 24h SFABR cycle, although initial rate was decreased. This indicates the suitability of SFABR configuration for the treatment of azo-dye containing wastewaters in the presence of nitrate.

Studies were carried out on 4-aminobenzenesulfonate (4-ABS) degradation by free and alginate entrapped cells of Agrobacterium sp. PNS-1. Degradation rate in batch reactors with free cells was marginally higher than Ca-encapsulated cells. Comparison of Ca2+ and Ba2+ as gelling agents showed that 4-ABS removal rate was significantly less with Ba-alginate entrapped cells. Specific degradation rates, using linear regression analysis and based on the initial biomass in the beads, varied from 49.7 mg/mg biomass/h to 92.0 mg/mg biomass/h for Ca-alginate encapsulated cells for different initial 4-ABS concentrations ranging from 200 to 800 mg/L. UV spectra of the aliquots drawn at different time intervals from batch reactors did not show accumulation of any intermediate during degradation. Ca-alginate immobilized cells could be repeatedly reused upto five cycles without any loss of activity. Studies with packed bed reactors, operated in a semi-continuous mode, showed that this could be used for 4-ABS degradation.

Yeast glutathione reductase is inactivated by pyridoxal 5'-phosphate (PLP). The reactivation of the enzyme by dilution as well as a characteristic absorption peak at 325 nm exhibited by NaBH4 - reduced - PLP modified enzyme show that the inactivation is due to the specific modification of the epsilon-amino group of lysine residue. The maximum of 70% inactivation was observed at 7 mM PLP and the equilibrium was reached within 3 min. Kinetic and equilibrium analysis of inactivation data derived at different PLP concentrations showed that a noncovalent intermediate is formed prior to inactivation. From the studies on the effect of pH on the inactivation rate, the pKa of epsilon-amino group of the reactive lysine residue was calculated to be 7.3. Among Various protecting agents tried, only NADP was found to be effective. The apparent stoichiometry of the reaction was one to one as the incorporation of 0.65 mole PLP/mole of enzyme led to 70% inactivation at saturating PLP concentration.

Yeast glutathione reductase was inactivated by the bifunctional reagent, o-phthalaldehyde. The initial rate of inactivation followed pseudo-first order kinetics. Fluorescence spectral properties of modified enzyme indicated the formation of an isoindole derivative from cysteine and lyaine residues present in close proximity as shown by typical fluorescence emission and excitation maximum at 410 nm and 337 nm, respectively. The fluorescence spectral studies with o-phthalaldehyde in the presence and absence of N-ethylmaleimide indicated that both the inhibitors react with the same cysteine residue, which is non-essential for enzyme activity. The coenzyme NADPH did not protect the enzyme against the o-phthalaldehyde reaction while oxidised glutathione prevented o-phthalaldehyde inactivation. This could be due to reaction of the amino group of glutathione with o-phthalaldehyde. Stoichiometry of the reaction showed that the formation of approximately 2 isoindole derivatives per subunit of glutathione reductase is accompanied by 75% loss of activity. The results suggest that o-phthalaldehyde binds to non-essential cysteine and lysine residues present in close proximity which results in conformational changes leading to enzyme inactivation.

Beef liver glutamate dehydrogenase (GDH) is inactivated by the bifunctional reagent, o-phthalaldehyde. The initial rate of inactivation follows pseudo first-order kinetics. The reaction of the enzyme with o-phthalaldehyde results in isoindole derivative formation which is characterized by typical fluorescence emission and excitation maximum at 410 nm and 337 nm, respectively. The inactivation of GDH by o-phthalaldehyde is partially prevented by alpha-ketoglutaric acid, whereas NADH does not provide any protection. This clearly indicates that cysteine and lysine residues are located near the alpha-ketoglutaric acid binding center. The dissociation constant of 2.2 mM was obtained for enzyme-alpha-ketoglutaric acid complex. Stoichiometry of o-phthalaldehyde binding with glutamate dehydrogenase showed that the formation of approximately one isoindole derivative per subunit of glutamate dehydrogenase is accompanied by complete loss of activity.

Yeast glutathione reductase was inactivated by pyridoxal 5'-phosphate. The inhibition was reversed by dilution. The enzyme-pyridoxal 5'-phosphate complex on reduction with sodium borohydride gave a characteristic absorption maximum at 325 nm and fluorescence maximum at 395 nm when exciated at 325 nm. These results were consistent with the reaction of epsilon-amino group of lysine residue of the enzyme with pyridoxal 5'-phosphate. The enzyme was protected against pyridoxal 5'-phosphate inhibition by NADP indicating thereby that the essential lysine residues are present during the NADP binding site.