Enzymatic reduction of azo and indigoid compounds

A customer- and environment-friendly method for the decolorization azo dyes was developed. Azoreductases could be used both to bleach hair dyed with azo dyes and to reduce dyes in vat dyeing of textiles. A new reduced nicotinamide adenine dinucleotide-dependent azoreductase of Bacillus cereus, which showed high potential for reduction of these dyes, was purified using a combination of ammonium sulfate precipitation and chromatography and had a molecular mass of 21.5 kDa. The optimum pH of the azoreductase depended on the substrate and was within the range of pH 6 to 7, while the maximum temperature was reached at 40°C. Oxygen was shown to be an alternative electron acceptor to azo compounds and must therefore be excluded during enzymatic dye reduction. Biotransformation of the azo dyes Flame Orange and Ruby Red was studied in more detail using UV-visible spectroscopy, high-performance liquid chromatography, and mass spectrometry (MS). Reduction of the azo bonds leads to cleavage of the dyes resulting in the cleavage product 2-amino-1,3 dimethylimidazolium and N∼1∼,N∼1∼-dimethyl-1,4-benzenediamine for Ruby Red, while only the first was detected for Flame Orange because of MS instability of the expected 1,4-benzenediamine. The azoreductase was also found to reduce vat dyes like Indigo Carmine (C.I. Acid Blue 74). Hydrogen peroxide (H₂O₂) as an oxidizing agent was used to reoxidize the dye into the initial form. The reduction and oxidation mechanism of Indigo Carmine was studied using UV-visible spectroscopy.     

Properties of NAD (P) H Azoreductase from Alkaliphilic Red Bacteria Aquiflexum sp. DL6

Azoreductase plays a key role in bioremediation and biotransformation of azo dyes. It initializes the reduction of azo bond in azo dye metabolism under aerobic or anaerobic conditions. In the present study, we isolated an alkaliphilic red-colored Aquiflexum sp. DL6 bacterial strain and identified by 16S rRNA method. We report nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate-dependent azoreductase purified from Aquiflexum sp. DL6 by a combination of ammonium sulfate precipitation and chromatography methods. The azoreductase was purified up to 30-fold with 37 % recovery. The molecular weight was found to be 80 kDa. The optimum activity was observed at pH 7.4 and temperature 60 °C with amaranth azo dye as a substrate. The thermal stability of azoreductase was up to 80 °C. The azoreductase has shown a wide range of substrate specificity, including azo dyes and nitro aromatic compounds. Metal ions have no significant inhibitory action on azoreductase activity. The apparent K _m and V _max values for amaranth azo dye were 1.11 mM and 30.77 U/mg protein respectively. This NAD (P) H azoreductase represents the first azoreductase to be characterized from alkaliphilic bacteria.     

Degradation of azo dyes containing aminonaphthol by Sphingomonas sp strain 1CX

Sphingomonas sp strain 1CX was isolated from a wastewater treatment plant and is capable of aerobically degrading a suite of azo dyes, using them as a sole source of carbon and nitrogen. All azo dyes known to be decolorized by strain 1CX (Orange II, Acid Orange 8, Acid Orange 10, Acid Red 4, and Acid Red 88) have in their structure either 1-amino-2-naphthol or 2-amino-1-naphthol. In addition, an analysis of the structures of the dyes degraded suggests that there are certain positions and types of substituents on the azo dye which determine if degradation will occur. Growth and dye decolorization occurs only aerobically and does not occur under fermentative or denitrification conditions. The mechanism by which 1CX decolorizes azo dyes appears to be through reductive cleavage of the azo bond. In the case of Orange II, the initial degradation products were sulfanilic acid and 1-amino-2-naphthol. Sulfanilic acid, however, was not used by 1CX as a growth substrate. The addition of glucose or inorganic nitrogen inhibited growth and decoloration of azo dyes by 1CX. Attempts to grow the organism on chemically defined media containing several different amino acids and sugars as sources of nitrogen and carbon were not successful. Phylogenetic analysis of Sphingomonas sp strain 1CX shows it to be related to, but distinct from, other azo dye-decolorizing Sphingomonas spp strains isolated previously from the same wastewater treatment facility. Received 19 May 1999/ Accepted in revised form 11 August 1999     

Correlation of anaerobic biodegradability and the electrochemical characteristic of azo dyes

Some experiments were conducted to study some electrochemical factors affecting the bacterial reduction (cleavage) of azo dyes, knowledge of which will be useful in the wastewater treatments of azo dyes. A common mixed culture was used as a test organism and the reductions of Acid Yellow 4, 11, 17 and Acid Yellow BIS were studied. It was found that the azo dyes were reduced at different rates, which could be correlated with the reduction potential of the azo compounds in cyclic voltammetric experiments. Acid Yellow BIS (E _r − 616.75 mV) was reduced at the highest rate of 0.0284 mol g dry cell weight⁻¹ h⁻¹, Acid Yellow 11 (E _r − 593.25 mV) at 0.0245 mol g dry cell weight⁻¹ h⁻¹ and Acid Yellow 4 (E _r − 513 mV) at 0.0178 mol g dry cell weight⁻¹ h⁻¹. At the same time, the decolourization rate of Acid Yellow 17 (E _r − 627.5 mV) was 0.0238 mol g dry cell weight⁻¹ h⁻¹, which was affected by the nature of chlorine substituent. Reduction of these azo dyes did not occur under aeration conditions. These studies with a common mixed culture indicate that the reduction of azo dyes may be influenced by the chemical nature of the azo compound. The reduction potential is a preliminary tool to predict the decolourization capacity of oxidative and reductive biocatalysts.     

Characterisation of the flavin-free oxygen-tolerant azoreductase from Xenophilus azovorans KF46F in comparison to flavin-containing azoreductases

The flavin-free azoreductase from Xenophilus azovorans KF46F (AzoB), which has been the very first characterized oxygen-tolerant azoreductase, was analyzed in comparison to various recently described flavin-containing azoreductases from different bacterial sources. Sequence comparisons demonstrated that the azoreductase from X. azovorans KF46F is a member of the NmrA family of proteins and demonstrates 30% sequence identity with a NADPH-dependent quinone oxidoreductase from Escherichia coli (encoded by ytfG). In contrast, it was found that the flavin-containing azoreductases from E. coli OY1-2 (AZR), Bacillus sp. OY1-2 (AZR) and related azoreductases all belong to the FMN_red superfamily of enzymes. The substrate specificity of AzoB was reanalyzed in respect to the recently characterized flavin-containing azoreductases, and it was found that purified AzoB converted in addition to different ortho-hydroxy azo compounds [such as Orange II = 1-(4′-sulfophenylazo)-2-naphthol] also the simple non-hydroxylated non-sulfonated azo dye Methyl Red (4′-dimethylaminoazobenzene-2-carboxylic acid), but no indications for the conversion of quinones were obtained. Significant differences were observed in the substrate specificities between AzoB and the flavin-containing azoreductases. The kinetic analysis of the turn-over of Orange II by AzoB suggested an ordered bireactant reaction mechanism which was different from the ping-pong mechanism suggested for the flavin-containing azoreductases.     

Characterization of thermostable FMN-dependent NADH azoreductase from the moderate thermophile Geobacillus stearothermophilus

The gene encoding an FMN-dependent NADH azoreductase, AzrG, from thermophilic Geobacillus stearothermophilus was cloned and functionally expressed in recombinant Escherichia coli. Purified recombinant AzrG is a homodimer of 23 kDa and bore FMN as a flavin cofactor. The optimal temperature of AzrG was 85 °C for the degradation of Methyl Red (MR). AzrG remained active for 1 h at 65 °C and for 1 month at 30 °C, demonstrating both superior thermostability and long-term stability of the enzyme. AzrG efficiently decolorized MR, Ethyl Red at 30 °C. Furthermore, the enzyme exhibited a wide-range of degrading activity towards several tenacious azo dyes, such as Acid Red 88, Orange I, and Congo Red. These results suggested the sustainable utilization of G. stearothermophilus as an azo-degrading strain for AzrG carrying whole-cell wastewater treatments for azo pollutants under high temperature conditions.     

Use of azo dye ligand chromatography for the partial purification of a novel extracellular peroxidase from Streptomyces viridosporus T7A

Crude peroxidase preparations from the lignocellulose-degrading actinomycete, Streptomyces viridosporus T7A, were shown to decolorize several azo dye isomers and showed a correlation of dye structure to degradability similar to that shown by fungal Mn-peroxidase, an enzyme not previously described in actinomycetes. Addition of the heme-peroxidase inhibitor KCN did not significantly change the ability of the T7A enzyme(s) to decompose the dyes. These results suggest that T7A may produce a Mn- or other peroxidase with similar substrate specificity to Mn-peroxidase. Affinity chromatography using immobilized azo dye isomers was used for purifying peroxidases from T7A. A significantly purified peroxidase preparation was obtained irrespective of the azo dye used. In comparison, concanavalin A lectin affinity chromatography showed very poor binding and resolution for T7A peroxidases. Azo dye affinity purification gave preparations sufficiently purified to allow amino acid microsequencing for two of the bound proteins. N-terminal amino acid sequences were found to share significant homology with a fungal Mn-peroxidase and actinomycete cellulases. Received: 20 May 1997 / Received revision: 17 December 1997 / Accepted: 2 January 1998     

Bacterial Decolorization of Azo Dyes by Rhodopseudomonas palustris

Summary The ability of Rhodopseudomonas palustris AS1.2352 possessing azoreductase activity to decolorize azo dyes was investigated. It was demonstrated that anaerobic conditions were necessary for bacterial decolorization, and the optimal pH and temperature were pH 8 and 30–35 °C, respectively. Decolorization of dyes with different molecular structures was performed to compare their degradability. The strain could decolorize azo dye up to 1250 mg l⁻¹, and the correlation between the specific decolorization rate and dye concentration could be described by Michaelis–Menten kinetics. Long-term repeated operations showed that the strain was stable and efficient during five runs. Cell extracts from the strain demonstrated oxygen-insensitive azoreductase activity in vitro.     

Decolorization of azo dyes by <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> MR-1 in the presence of humic acids

The effects of humic acid (HA) on azo dye decolorization by Shewanella oneidensis MR-1 were studied. It was found that HA species isolated from different sources could all accelerate the decolorization of Acid Red 27 (AR27). Anoxic and anaerobic conditions were required for the enhancement of azo dye decolorization by HA. In the presence of 50 mg DOC L⁻¹ Aldrich HA, 15–29% increases in decolorization efficiencies of azo dyes with different structures were achieved in 11 h. The enhancing effects increased with the increase of HA concentrations ranging from 25 to 150 mg DOC L⁻¹, and the decolorization rates were directly proportional to the HA concentrations when they were below 100 mg DOC L⁻¹. Lactate and formate were good electron donors for AR27 decolorization in the presence of HA. Both nitrate (0.1–3.0 mM) and nitrite (0.3–1.2 mM) inhibited AR27 decolorization in the presence of HA, and negligible decolorization was observed before their removal. Soluble FeCl₃ could accelerate the decolorization process in the presence of HA, whereas insoluble hematite could not. These findings may affect the understanding of bioremediation of azo dye-polluted environments and help improve the treatment of azo dye wastewaters.     

Identification, Isolation and characterization of a novel azoreductase from Clostridium perfringens

Azo dyes are used widely in the textile, pharmaceutical, cosmetic and food industries as colorants and are often sources of environmental pollution. There are many microorganisms that are able to reduce azo dyes by use of an azoreductase enzyme. It is through the reduction of the azo bonds of the dyes that carcinogenic metabolites are produced thereby a concern for human health. The field of research on azoreductases is growing, but there is very little information available on azoreductases from strict anaerobic bacteria. In this study, the azoreductase gene was identified in Clostridium perfringens, a pathogen that is commonly found in the human intestinal tract. C. perfringens shows high azoreductase activity, especially in the presence of the common dye Direct Blue 15. A gene that encodes for a flavoprotein was isolated and expressed in Escherichia coli, and further purified and tested for azoreductase activity. The azoreductase (known as AzoC) was characterized by enzymatic reaction assays using different dyes. AzoC activity was highest in the presence of two cofactors, NADH and FAD. A strong cofactor effect was shown with some dyes, as dye reduction occurred without the presence of the AzoC (cofactors alone). AzoC was shown to perform best at a pH of 9, at room temperature, and in an anaerobic environment. Enzyme kinetics studies suggested that the association between enzyme and substrate is strong. Our results show that AzoC from C. perfringens has azoreductase activity.     

Decolorization of the azo dye Acid Red 18 by crude manganese peroxidase: Effect of system parameters and kinetic study

Abstract

To optimize operating conditions for the decolorization of the azo dye Acid Red 18 (AR18) by crude manganese peroxidase (MnP), some important factors affecting enzymatic decolorization were systematically investigated. Under the optimal enzyme reaction conditions, a decolorization efficiency of more than 82.3% was achieved after 60 min treatment. Furthermore, the manganese chelators, malate, tartrate, and lactate were found to be more favorable for the decolorization of AR18 than malonate, acetate, succinate, maleate, oxalate, and citrate. However, the presence of NaCl or Na₂SO₄ had a negative impact on the decolorization of AR18. The K_m and V_max values of MnP for AR18 were 169.66 μmol L^{? 1} and 20.63 μmol L^{? 1} min^{? 1}, respectively. The decolorization of AR18 by MnP followed second-order reaction kinetics with respect to the dye concentration. The decolorization rate constant increased with increasing temperature from 20°C to 35°C, which indicated an activation energy (E_a) of 15.87 kcal mol^{? 1} and frequency factor (k₀) of 1.36 × 10⁸ mg^{? 1} L min^{? 1} according to the Arrhenius equation. The results obtained provide experimental data for the application of crude MnP for the decolorization of AR18, and help to elucidate the biochemical mechanism of dye decolorization by the enzyme.     

Aerobic biodegradation of a mixture of sulfonated azo dyes by a bacterial consortium immobilized in a two‐stage sparged packed‐bed biofilm reactor

The biodegradation of the sulfonated azo dyes, Acid Orange 7 (AO7) and Acid Red 88 (AR88), by a bacterial consortium isolated from water and soil samples obtained from sites receiving discharges from textile industries, was evaluated. For a better removal of azo dyes and their biodegradation byproducts, an aerobically operated two‐stage rectangular packed‐bed biofilm reactor (2S‐RPBR) was constructed. Because the consortium's metabolic activity is affected by oxygen, the effect of the interstitial air flow rate Q_GI on 2S‐RPBR's zonal values of the oxygen mass transfer coefficient k_La was estimated. In the operational conditions probed in the bioreactor, the k_La values varied from 3 to 60 h^?1, which roughly correspond to volumetric oxygen transfer rates, dc_L/dt, ranging from 20 to 375 mg O₂ L^?1h^?1. Complete biodegradation of azo dyes was attained at loading rates B_V,AZ up to 40 mg L^?1d^?1. At higher B_V,AZ values (80 mg L^?1 d^?1), dye decolorization and biodegradation of the intermediaries 4‐amino‐naphthalenesulphonic acid (4‐ANS) and 1‐amino‐2‐naphthol (1‐A2N) was almost complete. However, a diminution in COD and TOC removal efficiencies was observed in correspondence to the 4‐aminobenzenesulfonic acid (4‐ABS) accumulation in the bioreactor. Although the oxygen transport rate improved the azo dye mineralization, the results suggest that the removal efficiency of azo dyes was affected by biofilm detachment at relatively high Q_GI and B_V,AZ values. After 225 days of continuous operation of the 2S‐RFBR, eight bacterial strains were isolated from the biofilm attached to the porous support. The identified genera were: Arthrobacter, Variovorax, Agrococcus, Sphingomonas, Sphingopyxis, Methylobacterium, Mesorhizobium, and Microbacterium.     

Decolorization of triphenylmethane, azo, and anthraquinone dyes by a newly isolated Aeromonas hydrophila strain

A broad-spectrum dye-decolorizing bacterium, strain DN322, was isolated from activated sludge of a textile printing wastewater treatment plant. The strain was characterized and identified as a member of Aeromonas hydrophila based on Gram staining, morphology characters, biochemical tests, and nearly complete sequence analysis of 16S rRNA gene and the gyrase subunit beta gene (gyrB). Strain DN322 decolorized a variety of synthetic dyes, including triphenylmethane, azo, and anthraquinone dyes. For color removal, the most suitable pH and temperature were pH 5.0–10.0 and 25–37°C, respectively. Triphenylmethane dye, e.g., Crystal Violet, Basic Fuchsin, Brilliant Green, and Malachite Green (50 mg l⁻¹) were decolorized more than 90% within 10 h under aerobic culture condition and Crystal Violet could be used as sole carbon source and energy source for cell growth. The color removal of triphenylmethane dyes was due to a soluble cytosolic enzyme, and the enzyme was an NADH/NADPH-dependent oxygenase; For azo and anthraquinone dyes, e.g., Acid Amaranth, Great Red GR, Reactive Red KE-3B, and Reactive Brilliant Blue K-GR (50 mg l⁻¹) could be decolorized more than 85% within 36 h under anoxic condition. This strain may be useful for bioremediation applications.     

Decolorization of Acid Red 27 and Reactive Red 2 by <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> under a batch system

The objectives of this study were to investigate: (1) the capacity of Enterococcus faecalis on the decolorization of the azo dyes Acid Red 27 and Reactive Red 2; and (2) the growth characteristics of E. faecalis on those dyes. E. faecalis was able to decolorize Acid Red 27 and Reactive Red 2 effectively. High decolorization efficiency (95–100%) was achieved within 3 h of incubation for Acid Red 27, and 12 h for Reactive Red 2, at room temperature, neutral pH, static and non-aerated condition. Growth characteristics of E. faecalis on azo dyes, which were indicated by cell growth rate, biomass production, and growth yield, was worse than the control. E. faecalis grew better on Acid Red 27 rather than Reactive Red 2.     

Enhancing the adsorption capacities of acid dyes by chitosan nano particles

In the present study, nanochitosan emulsion has been produced in a suspension form by adding tripolyphosphate solution into a chitosan solution drop-wise. The adsorption capacities of four acid dyes, namely, Acid Orange 10 (AO10), Acid Orange 12 (AO12), Acid Red 18 (AR18) and Acid Red 73 (AR73) on nanochitosan, have been determined to be 1.77, 4.33, 1.37 and 2.13 mmol l(-1), respectively. The nanochitosan dye capacities were compared with normal chitosan capacities which were 1.54, 2.66, 1.11 and 1.25 mmol l(-1) for AO10, AO12, AR18 and AR73, respectively. In all cases, the nanochitosan has a higher capacity. The mechanism of acid dye adsorption and the effect of pH are also discussed.     

Intraparticle diffusion processes during acid dye adsorption onto chitosan

The adsorption of five acid dyes onto chitosan was studied. The equilibrium capacities based on the Langmuir analysis were 1.54, 2.66, 1.11, 1.25 and 1.03 mmol/g chitosan for Orange 10 (AO10), Acid Orange 12 (AO12), Acid Red 18 (AR18), Acid Red 73 (AR73) and Acid Green 25 (AG25) respectively. The batch adsorption rate for the five systems based on an intraparticle diffusion rate parameter derived from the plots of dye adsorbed versus the square root of time indicated that the adsorption mechanism was predominantly intraparticle diffusion but there was also a dependence on pore size as the dye diffuses through macropore, mesopore and micropore respectively.     

Molecular determinants of azo reduction activity in the strain <Emphasis Type="Italic">Pseudomonas putida</Emphasis> MET94

Azo dyes are the major group of synthetic colourants used in industry and are serious environmental pollutants. In this study, Pseudomonas putida MET94 was selected from 48 bacterial strains on the basis of its superior ability to degrade a wide range of structurally diverse azo dyes. P. putida is a versatile microorganism with a well-recognised potential for biodegradation or bioremediation applications. P. putida MET94 removes, in 24 h and under anaerobic growing conditions, more than 80% of the majority of the structurally diverse azo dyes tested. Whole cell assays performed under anaerobic conditions revealed up to 90% decolourisation in dye wastewater bath models. The involvement of a FMN dependent NADPH: dye oxidoreductase in the decolourisation process was suggested by enzymatic measurements in cell crude extracts. The gene encoding a putative azoreductase was cloned from P. putida MET94 and expressed in Escherichia coli. The purified P. putida azoreductase is a 40 kDa homodimer with broad substrate specificity for azo dye reduction. The presence of dioxygen leads to the inhibition of the decolourisation activity in agreement with the results of cell cultures. The kinetic mechanism follows a ping-pong bi–bi reaction scheme and aromatic amine products were detected in stoichiometric amounts by high-performance liquid chromatography. Overall, the results indicate that P. putida MET94 is a promising candidate for bioengineering studies aimed at generating more effective dye-reducing strains.     

Monoazo and diazo dye decolourisation studies in a methanogenic UASB reactor

Mixed anaerobic bacterial consortia have been show to reduce azo dyes and batch decolourisation tests have also demonstrated that predominantly methanogenic cultures also perform azo bond cleavage. The anaerobic treatment of wool dyeing effluents, which contain acetic acid, could thus be improved with a better knowledge of methanogenic dye degradation. Therefore, the decolourisation of two azo textile dyes, a monoazo dye (Acid Orange 7, AO7) and a diazo dye (Direct Red 254, DR254), was investigated in a methanogenic laboratory-scale Upflow Anaerobic Sludge Blanket (UASB), fed with acetate as primary carbon source. As dye concentration was increased a decrease in total COD removal was observed, but the acetate load removal (90%) remained almost constant. A colour removal level higher than 88% was achieved for both dyes at a HRT of 24h. The identification by HPLC analysis of sulfanilic acid, a dye reduction metabolite, in the treated effluent, confirmed that the decolourisation process was due mainly to azo bond reduction. Although, HPLC chromatograms showed that 1-amino-2-naphthol, the other AO7 cleavage metabolite, was removed, aeration batch assays demonstrated that this could be due to auto-oxidation and not biological mineralization. At a HRT of 8h, a more extensive reductive biotransformation was observed for DR254 (82%) than for AO7 (56%). In order to explain this behaviour, the influence of the dye aggregation process and chemical structure of the dye molecules are discussed in the present work.     

Oxidative degradation of Amaranth dye by a new genus Kerstersia sp.

A dye-decolorizing bacterium was isolated from a coconut coir sample and identified as a new genus Kerstersia sp. by various biochemical tests and 16S rRNA gene sequencing. This bacterium was capable of degrading sulfonated azo dye Amaranth aerobically at 40?°C and pH 7.0. Tests conducted on intracellular crude enzyme extract identified an oxygen insensitive azoreductase. The optimum dye-decolorizing activity at pH 7.0 and 40?°C for the decolorization of dye was 0.091?U mL^?1 (μ_max 0.522?mg h^?1). The K_s 104.51?μM^?1 has been evaluated by plotting Lineweaver–Burk plot for the Amaranth dye. The dye degraded products were extracted and characterized by TLC, diazotization and Carbylamines test, which indicated that Amaranth was biotransformed into non-toxic aromatic metabolite without amine group.