Sulphonated Azo Dye Research Articles

Leveraging molecular docking to understand Congo red degradation by Staphylococcus caprae MB400.

Congo red (CR) is a genotoxic, sulphonated azo dye and poses significant pollution problem. We hereby report its degradation by Staphylococcus caprae MB400. The bacterium initially propagated as a suspected contaminant upon CR dye supplemented nutrient agar plates, forming zones of clearance around its growth area. The bacterium was purified, gram stained and identified as Staphylococcus caprae via 16S rRNA gene sequencing. Dye decolourization was analysed in liquid culture, and Fourier-transform infrared spectroscopy (FTIR) was conducted for analysis of degraded product/metabolites. A decolourization of ~ 96.0% at 100µg/ml concentration and pH 7 after 24h of incubation was observed. Structure of the azoreductase enzyme, responsible for breakage of the bond in the dye and ultimately decolourization, was predicted, and molecular docking was harnessed for understanding the mechanism behind the reduction of azo bond (-N=N-) and conversion to metabolites. Our analysis revealed 12 residues critical for structural interaction of the azoreductase enzyme with this dye. Among these, protein backbone region surrounding four residues, i.e. Lys65, Phe122, Ile166 and Phe169, showed major displacement changes, upon binding with the dye. However, overall the conformational changes were not large.

Analysis of the azoreductase gene harbored by Alcaligenes sp. YB4 capable of concurrent removal of sulphonated azo dye and hexavalent chromium

Analysis of the azoreductase gene harbored by Alcaligenes sp. YB4 capable of concurrent removal of sulphonated azo dye and hexavalent chromium

Sulphonated azo dye decolorization by Alcaligenes faecalis subsp. phenolicus MB207: Insights from laboratory and computational analysis

Microbial decolorization of azo dyes, mediated by an enzymatic mechanism is an intricate cost-effective, and eco-friendly treatment method of genotoxic azo pollutants. Scientists are on the constant lookout for microbes, enzymes, and mechanisms that could aid remediation of the environment at a fast pace. Alcaligenes faecalis subsp. phenolicus MB207 is one such bacteria, consisting of azoreductase (AzoR) and laccase/multicopper oxidase enzyme responsible for sulphonated mono-azo dye (Methyl orange) and di-azo dye (Congo red) degradation. AzoR degrades dyes by a ping-pong setup while multicopper oxidase achieves this through a non-specified radical approach. We have coupled experimental analysis with bioinformatics for deciphering intricacies of this procedure in tiny scale enzymatic machines of this biotope. The degradation assays were followed by molecular docking of the enzyme-substrate complexes. Key anchoring bonds were detected and mapped H-bonding, electron exchange, ionic interactions, as well as hydrophobic interactions, provided insights into dye-enzyme and NADH-enzyme binding. This study establishes a foundation of the molecular basis of dye interaction with azoR and multicopper oxidase in A. facealis subsp. phenolicus MB207.

Enzymatic degradation of sulphonated azo dye using purified azoreductase from facultative Klebsiella pneumoniae.

Heterologously expressed and purified azoreductase enzyme from facultative Klebsiella pneumoniae was used to degrade sulphonated azo dye. Methyl orange (MO) was used as the model dye to study the azo dye decolorization potential of the purified enzyme at different conditions. The enzyme had maximum activity at 40 °C and pH 8.0. The enzyme was observed to be thermo-stable as some enzyme activity was retained even at 80 °C. The apparent kinetic parameters, i.e., appKm and appVmax, for azoreductase using MO as a substrate were found to be 17.18 μM and 0.08/min, respectively. The purified enzyme was able to decolorize approximately 83% of MO (20 μM) within 10 min in the presence of NADH. Thus, efficient decolorization of MO was observed by the purified enzyme. The recombinant enzyme was purified approximately 18-fold with 46% yield at the end of four steps of the purification process. Enzyme was present in a tetrameric structure as confirmed by the volume at which protein was eluted in gel filtration chromatography, and the monomeric molecular mass of enzyme was found to be 23 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The dye degradation efficiency of azoreductase cloned from Klebsiella pneumoniae and purified from recombinant Escherichia coli was observed to be much higher as compared with the efficiencies of the reported azoreductases from other bacterial strains. In the present study, we report the purification and characterization of the azoreductase cloned from Klebsiella pneumoniae and expressed in Escherichia coli.

Sustainable approach to decolourize methyl orange dye from aqueous solution using novel bacterial strain and its metabolites characterization

The present report deals with the identification of new bacteria for the degradation of sulphonated azo dye such as methyl orange because this type of dye is difficult to degrade by the physico-chemical method. Hence, an eco-friendly and efficient technique like biological method was studied to degrade methyl orange. Initially, three strains were isolated from the flasks containing media amended with textile wastewater using the serial dilution process. Out of three strains, micrococcus yunnanensis exhibited a higher decolourization efficacy in 100 mg L−1, and hence, this strain was chosen for further optimization studies and metabolites identification. This is the first report to study the micrococcus species in methyl orange degradation according to our knowledge. The optimum parameters for the decolourization of methyl orange were found to be pH 7, temperature at 30 °C and dye concentration at 100 mg L−1. The carbon source and nitrogen source suited well for the decolourization were observed as 1% glucose and 1% peptone. The metabolites obtained after degradation of methyl orange were analysed using FT-IR and GC–MS analysis. The final product obtained from the degraded sample was found to be N, N-Diethyl-N’-methyl-1,3-propane diamine.

Efficient decolorization and detoxification of sulphonated azo dye Ponceau 4R by using single and mixed bacterial consortia

The decolorization of toxic azo dye Ponceau 4R by three strains of bacteria Bacillus sp. strain AK1, Lysinibacillus sp. strain AK2 and Kerstersia sp. strain VKY1 individually and in consortia was studied. At optimal conditions, up to 95%, 93% and 87% of the dye was decolorized by the strains AK1, AK2 and VKY1, respectively, in 24 h at 200 mg/L of the dye. Decolorization of the dye was optimized for different parameters such as the concentration of dye, pH, temperature and NaCl concentration. These strains were able to decolorize Ponceau 4R up to an initial concentration of 800 mg/L in the pH range of 5–10, temperature 25–55 °C and NaCl concentration up to 30 g/L. The dye decolorization efficiency of these strains was further enhanced by using different consortia of AK1, AK2 and VKY1 in various combinations. The complete decolorization of the dye by a consortium was achieved within 18 h at 200 mg/L. The cell-free extract of these strains grown on this dye exhibited a remarkable activity of azoreductase which is involved in the breakage of the azo bond. The steady-state kinetics of azoreductase, validated the ping pong Bi-Bi mechanism of enzyme action. UV–Vis spectra, HPLC, FTIR and LC-MS analysis of the dye decolorized samples showed the formation of 4-aminonaphthalene-1-sulphonic acid and 5-amino-6-hydroxynaphthalene-2, 4-disulphonic acid as the products of azo bond breakage. The phytotoxicity test of decolorized sample revealed a considerable reduction in the toxicity in comparison with the parent dye.

Optimization of Factors Affecting Decolourization of Sulphonated Azo Dye Red HE7b in vitro by bacillus Sp. Azo1

A zo dyes are widely used organic molecules that are generally recalcitrant to biodegradation, thus causing environmental pollution. In this study, the effect of various growth conditions like temperature, pH, dye concentration, aeration and presence/absence of co-substrate on decolourization of azo dye Red HE7B by Bacillus sp. Azo1 has been determined. Decolourization of the dye was achieved under static condition. Optimum decolourization of Red HE7B was observed at 30°C and 50 mg/l dye concentration, where more than 90% decolourization was achieved within three days of inoculation. Bacillus sp. Azo1 could decolourize the dye through a range of pH values from 6 to 9. Presence of co-substrate limited the decolourization with glucose being the most potential inhibitor. Identification of factors influencing decolourization will help in rapid removal of these pollutants from contaminated habitat.

Degradation of reactive blue 13 using hydrodynamic cavitation: Effect of geometrical parameters and different oxidizing additives

Decolorization of reactive blue 13 (RB13), a sulphonated azo dye, was investigated using hydrodynamic cavitation (HC). The aim of research article is to check the influence of geometrical parameters (total flow area, the ratio of throat perimeter to its cross-sectional area, throat shape and size, etc.) and configuration of the cavitating devices on decolorization of RB13 in aqueous solution. For this purpose, eight cavitating devices i.e. Circular and slit venturi, and six orifice plates having different flow area and perimeter were used in the present work. Initially, the effects of various operating parameters such as solution pH, initial dye concentration, operating inlet pressure and cavitation number on the decolorization of RB13 have been investigated, and the optimum operating conditions were found. Kinetic analysis revealed that the decolorization and mineralization of RB13 using HC followed first order reaction kinetics. Almost 47% decolorization of RB13 was achieved using only HC with slit venturi as a cavitating device at an optimum inlet pressure of 0.4MPa and pH of the solution as 2.0. It has been found that in case of orifice plates, higher decolorization rate of 4×10−3min−1 was achieved using orifice plate 2 (OP2) which is having higher flow area and perimeter (α=2.28). The effect of process intensifying agents (hydrogen peroxide and ferrous sulphate) and different gaseous additives (oxygen and ozone) on the extent of decolorization of RB13 were also examined. Almost 66% decolorization of RB13 was achieved using HC combined with 2Lmin−1 of oxygen and in combination with ferrous sulphate (1:3). Nearly 91% decolorization was achieved using HC combined with H2O2 at an optimum molar ratio (dye:H2O2) of 1:20 while almost complete decolorization was observed in 15min using a combination of HC and ozone at 3gh−1 ozone feed rate. Maximum 72% TOC was removed using HC coupled with 3gh−1 ozone feed rate.

Evaluating role of immobilized periphyton in bioremediation of azo dye amaranth

The aim of this study was to evaluate the bioremediation capabilities of three kinds of periphyton (i.e. epiphyton, metaphyton and epilithon) immobilized in bioreactors to decolorize and biodegrade the sulphonated azo dye, amaranth. Results showed that periphyton dominated by phyla including Cyanobacteria, Proteobacteria and Bacteroidetes. Complete removal of dye was shown by all the biofilms periphyton (epiphyton showed highest removal efficacy) over a range of initial concentrations (50–500mgL−1) within 84h at pH 7 and 30°C. Biodegradation of amaranth was confirmed through FTIR and HPLC and the biodegradation pathways were detected by GC–MS/MS analysis. The azo bonds in the amaranth were successfully broken by periphyton and amaranth was converted to non-toxic, aliphatic compounds including isobutene, acetyl acetate and ethyl acetate. The results showed the potential application of immobilized periphyton at industrial scale for the removal of azo dyes from wastewater containing azo dye amaranth.

Periphyton biofilms: A novel and natural biological system for the effective removal of sulphonated azo dye methyl orange by synergistic mechanism

Due to their large scale use, azo dyes are adversely affecting aquatic fauna and flora as well as humans. The persistent nature of sulphonated azo dyes makes them potential ecotoxic hazards. The aim of the present study was to employ a proficient, locally available biomaterial, viz. periphyton (i.e. epiphyton, epilithon or metaphyton), for removal of the azo dye, methyl orange (MO). Results showed that the periphytic biofilms are capable of completely removing comparatively high concentrations (up to 500 mg L−1) of MO from wastewater. The removal of MO occurs by a synergistic mechanism involving bioadsorption and biodegradation processes. The adsorption of MO by periphyton can be described by pseudo-second order kinetics. Elovich and intraparticle diffusion models as well as Langmuir equations fit well to the MO adsorption process. FTIR analysis of MO and its metabolites demonstrated biotransformation into simpler compounds within 72 h. GC-MS/MS analysis showed the conversion of MO into simpler compounds such as phenol, ethyl acetate and acetyl acetate. The results indicated that periphyton is a promising biomaterial for the complete removal of MO from wastewater and that the treatment process has the potential for in situ removal of MO at contaminated sites.

Effect of ZnO nanoparticles on biodegradation and biotransformation of co-substrate and sulphonated azo dye in anaerobic biological sulfate reduction processes

In recent years, rapid increase of nanomaterial applications exposed the wastewater treatment plants to the new challenges. Potential effects of nanoparticles (NPs) on biological sulfate reduction and dyes biodegradation in biological wastewater treatment systems still need to be investigated. This study explored the effects of zinc oxide nanoparticles (ZnO-NPs) on sulfate reducing bacteria (SRB) culture treating textile wastewater. Batch reactor studies were performed by exposing the mixed SRB culture to different initial ZnO-NPs concentrations. Increasing ZnO-NPs concentrations from 0 to 200 mg l−1 inhibited the reactor performance by decreasing COD, sulfate, and color removal efficiencies from 54.4%, 78.1%, and 95.4% to 30.1%. 44.0%, and 80.0%, respectively. Moreover, dye biotransformation was also adversely affected in the presence of ZnO-NPs as revealed by the increase in total aromatic amines formation and the decrease in activities of various oxido-reductive enzymes such as veratryl alcohol oxidase, lignin peroxidase, and azo reductase.

Correction: Bacterial–yeast consortium as an effective biocatalyst for biodegradation of sulphonated azo dye Reactive Red 198

Correction for ‘Bacterial–yeast consortium as an effective biocatalyst for biodegradation of sulphonated azo dye Reactive Red 198’ by Mayur B. Kurade et al., RSC Adv., 2015, 5, 23046–23056.

Bacterial–yeast consortium as an effective biocatalyst for biodegradation of sulphonated azo dye Reactive Red 198

A novel bacterial–yeast consortium (Brevibacillus laterosporusandGalactomyces geotrichum) acts as a proficient biocatalyst.

Development of low cost upflow column bioreactor for degradation and detoxification of Blue HERD and textile effluent by Lysinibacillus sp. RGS immobilized on Loofa

Dyes and other toxic components of textile wastewater create severe hazards to receiving water. In the present study, Lysinibacillus sp. RGS, an effective dye degrading bacteria immobilized on Luffa cylindrica (Loofa) was used to decolorize sulphonated azo dye Blue HERD and 50% real textile effluent in an upflow column bioreactor. Blue HERD (200 mg L−1) and 50% real textile effluent were degraded at volumetric flow rate 40 mL h−1 and 15 mL h−1, respectively. Induction in reductive (NADH–DCIP reductase and azoreductase) as well as oxidative (laccase, tyrosinase and veratryl alcohol oxidase) enzymes proved enzymatic decolorization of Blue HERD and 50% textile effluent. Significant reduction in COD, BOD, TOC, hardness and alkalinity of decolorized samples of Blue HERD and 50% effluent confirmed mineralization. HPTLC, FTIR and GCMS analysis confirmed degradation of Blue HERD into simple intermediates during decolorization in bioreactor. Less toxic nature of metabolites was confirmed using genotoxicity, cytotoxicity and phytotoxicity tests.

Microaerophilic degradation of sulphonated azo dye – Reactive Red 195 by bacterial consortium AR1 through co-metabolism

The textile and dye industries are considered as one of the foremost sectors that pollutes environment. Technologies employing biological methods showed promising approach to remediate sites polluted with dye and dye intermediates. Bacterial consortium AR1 developed through culture enrichment method was comprised of four distinct bacterial strains. The synergistic metabolic activities of AR1 led to complete decolourization of monoazo dye Reactive Red 195 within 14 h under microaerophilic environment. The co-metabolic decolourization of Reactive Red 195 by consortium, in presence of maltose and proteose peptone (0.1%, w/v, each) in minimal medium, easily reduced Reactive Red 195 (100 mg/L) for five consecutive cycles without any replenishment of nutrient. The maximum decolourization was observed at pH 8.0 and 40 °C. The consortium was acclimatized to decolourize Reactive Red 195 at higher concentration (2000 mg/L) even under high salt concentration (1 M NaCl). Consortium exhibited broad substrate specificity where it decolourized 15 structurally different dyes and more than 50% decolourization was observed in a medium containing mixture of dyes. The degradation products analyzed using FTIR, HPTLC and 1H NMR revealed the formation of 2-amino-naphthalene, 1-amino-benzene and 1-nitro-benzene. Thus the ability of bacterial consortium for simultaneous decolourization and degradation of azo compounds signifies its potential application in dye remediation.

Degradation of Sulphonated Azo Dye Red HE7B by Bacillus sp. and Elucidation of Degradative Pathways

Bacteria capable of degrading the sulfonated azo dye Red HE7B were isolated from textile mill effluent contaminated soil. The most efficient isolate was identified as Bacillus sp. Azo1 and the isolate could successfully decolorize up to 89% of the dye. The decolorized cultural extract analyzed by HPLC confirmed degradation. Enzymatic analysis showed twofold and fourfold increase in the activity of azoreductase and laccase enzymes, respectively, indicating involvement of both reductive and oxidative enzymes in biodegradation of Red HE7B. Degraded products which were identified by GC/MS analysis included various metabolites like 8-nitroso 1-naphthol, 2-diazonium naphthalene. Mono azo dye intermediate was initially generated from the parent molecule. This mono azo dye was further degraded by the organism, into additional products, depending on the site of cleavage of R-N=N-R molecule. Based on the degradation products identified, three different pathways have been proposed. The mechanism of degradation in two of these pathways is different from that of the previously reported pathway for azo dye degradation. This is the first report of a microbial isolate following multiple pathways for azo dye degradation. Azo dye Red HE7B was observed to be phytotoxic, leading to decrease in root development, shoot length and seedling fresh weight. However, after biotreatment the resulting degradation products were non-phytotoxic.

Evaluation of in vitro efficacy for decolorization and degradation of commercial azo dye RB-B by Morganella sp. HK-1 isolated from dye contaminated industrial landfill

Reactive Black-B (RB-B) – one of the multi-sulphonated reactive azo dye – is being used extensively in textile as well as paper industries. Reactive azo dyes comprise of a significant group of synthetic compounds categorized as xenobiotics and its abatement from the environment still remains a challenge. In the present study, a newly isolated indigenous bacterial strain Morganella sp. HK-1 was exploited for its ability to decolorize and degrade RB-B dye. The isolate completely degraded RB-B (20gL−1) within 24h under static conditions. Furthermore, the visible and FTIR spectral analysis established the bio-degradation of RB-B. The degraded metabolites of RB-B by Morganella sp. HK-1 were identified by GC–MS analysis as disodium 3,4,6-triamino-5-hydroxynaphthalene-2,7-disulfonate, 4-aminophenylsulfonylethyl hydrogen sulfate, naphthalene-1-ol, aniline and benzene. Based on this information, a putative pathway of degradation of RB-B by Morganella sp. HK-1 has been proposed. This study is the first report on elucidation of mechanism of bacterial degradation of RB-B dye. Furthermore, phytotoxicity, genotoxicity and aquatic acute toxicity studies of the parent dye and the bio-degraded dye products revealed drastic reduction in the toxicity of metabolites as compared to the parent dye. This implies that the biotreatment of the dye is of non-toxic nature. This study thus indicates the effectiveness of Morganella sp. HK-1 for the treatment of textile effluents containing sulphonated azo dyes.

Study of bio-degradation and bio-decolourization of azo dye by Enterobacter sp. SXCR

The objective of this study was to evaluate the decolourization potential of textile dyes by a relatively newly identified bacteria species, Enterobacter sp. SXCR which was isolated from the petroleum polluted soil samples. The bacterial strain was identified by 16S rRNA gene sequence analysis. The effects of operational conditions like initial dye concentration, pH, and temperature were optimized to develop an economically feasible decolourization process. The isolate was able to decolourize sulphonated azo dye (Congo red) over a wide range (0.1–1 gl−1), pH 5–9, and temperature 22–40°C in static condition. Anaerobic condition with minimal salt medium supplemented with 2 gl−1 glucose, pH 7 and 34°C were considered to be the optimum decolourizing condition. The bacterial isolate SXCR showed a strong ability to decolourize dye (0.2 gl−1) within 93 h. The biodegradation was monitored by UV–vis, fourier transform infra-red spectroscopy (FTIR) spectroscopy and high performance liquid chromatography (HPLC). Furthermore, the involvement of azoreductase in the decolourization process was identified in this strain. Cells of Enterobacter cloacae were immobilized by entrapment in calcium-alginate beads. Immobilized bacterial cells were able to reduced azo bonds enzymatically and used as a biocatalyst for decolourization of azo dye Congo red. Michaelis–Menten kinetics was used to describe the correlation between the decolourization rate and the dye concentration.

Decolorization and Degradation of Reactive Dye in Aqueous Solution by Ozonation in a Semi-batch Bubble Column Reactor

The decolorization and degradation of anionic sulphonated azo dye (Reactive orange 16 (RO16)), which is suspected to be carcinogenic, were investigated using ozone. The decolorization process of the reactive dye was carried out by bubbling ozone at the bottom of a bubble column reactor containing the dye solution. The effect of pH, reaction time, dye concentration, ozone concentration, and decolorization time was studied. Also, degradation products and possible degradation mechanism were investigated. The results showed that ozonation was a highly effective way to remove color from wastewater. The color of a synthetic waste solution containing water-soluble reactive dye was reduced to 69.69 % under the basic condition (pH 12), with complete RO16 degradation occurring in 8 min. Ozone consumption continued for a further 16 min after which time most of the degradation reactions were complete. Kinetic studies showed that direct ozonation of the aqueous dyes represented a pseudo-first-order reaction with respect to the dye. The apparent rate constant increased with both the applied ozone dose and higher pH values and declined logarithmically with the initial dye concentration. Intermediates such as 6-acetylamino-3-aminonaphthalene-2-sulfonic acid, 2-(4-nitrosophenyl) sulfonylethyl hydrogen sulfate, and 6-acetamido-4-hydroxy-3-nitroso naphthalene-2-sulfonic acid were detected by gas chromatograph coupled with mass spectrometry in the absence of pH buffer, while nitrate and sulfate ions and formic, acetic, and oxalic acids were detected by ion chromatography.

Response surface methodology mediated optimization of Remazol Orange decolorization in plain distilled water by Pseudomonas aeruginosa BCH

The bacterium Pseudomonas aeruginosa BCH decolorized and degraded the sulphonated azo dye Remazol Orange in plain distilled water. The effects of different parameters, i.e. pH, temperature and cell mass concentration on the biodegradation of dye in aqueous phase was evaluated using response surface methodology. Optimization was carried out using three-level Box–Behnken design. Predicted values were found to be in good agreement with experimental values (R 2 0.9997 and pred R 2 0.9984), which indicated suitability of the employed model and the success of response surface methodology. Optimum dye decolorization was maximized and the favourable conditions were pH 7.43, temperature 29.39 °C and cell mass concentration 2.88 g l−1, which resulted in 96.01 % decolorization within 5 h. It was validated from the predicted response (97.37 %). According to the analysis of variance results, the proposed model can be used to navigate the design space. 3D plot analysis disclosed the significant interaction between all three tested factors on decolorization process. The combinations of the three variables predicted during response surface methodology were confirmed through confirmatory experiments. Observations indicated that higher cell mass accelerated the decolorization process. Degradation of the dye was verified through high performance liquid chromatography analysis. Phytotoxicity studies carried out with dye and dye metabolites using Phaseolus mungo, Triticum aestivum and Sorghum vulgare indicated the detoxification of dye.