Investigation of isolation and immobilization of a microbial consortium for decoloring of azo dye 4BS

Eight high-effective decolorization strains were isolated by enrichment using Direct Fast Scarlet 4BS as sole source of carbon and energy. The optimal microbial consortium consisting of fungus 8-4(*) and bacterium 1-10 was selected by optimizing combination decolorization experiments with these eight freely suspended strains, whose decolorization activity was higher than individual strains due to synergistic reaction with each other. The optimal microbial consortium was also immobilized using polyvinyl alcohol (PVA) as the carrier. This paper optimized the immobilization and operational conditions, investigated the effect of the environmental factors (temperature, pH and dissolved oxygen (DO)) and initial dye concentration on the rate of decolorization by immobilized microbial consortium. The results showed that the optimal decolorization activity was observed in pH range (5-8), temperature range (25-40 degrees C) under shaking culture of high DO level. Decolorization with the optimal microbial consortium gave a relatively high maximum decolorization activity (ca. 81.25 mgl(-1)h(-1)), which occurred at a dye concentration of 1000 mgl(-1), suggesting the applicability of the strains in remediation of wastewater containing high azo dye concentrations. The immobilized beads could be reused for more than 30 cycles, without losing any degradation capacity. The changes of UV-visible spectra and the change curve of COD of 4BS solution before and after decolorization cultivation and the proliferation and distribution of microbial consortium in gel beads were also microscopically observed, which could be used for conferring the decolorization mechanisms of dye 4BS.     

Comparative studies on potential of consortium and constituent pure bacterial isolates to decolorize azo dyes

The decolorization potential of the consortium HM-4 constituted by mixing four laboratory isolates identified as Bacillus cereus (BN-7), Pseudomonas putida (BN-4), Pseudomonas fluorescens (BN-5) and Stenotrophomonas acidaminiphila (BN-3) was compared with that of individual isolates. Six different azo dyes viz., C.I. Acid Red 88 (AR-88), C.I. Acid Red 119 (AR-119), C.I. Acid Red 97 (AR-97), C.I. Reactive Red 120 (RR-120), C.I. Acid Blue 113 (AB-113) and C.I. Acid Brown 100 (AB-100) were used in this study. The individual bacterial isolates were not able to completely decolorize these dyes, except for dyes AR-119 and AB-113. The consortium HM-4 was able to decolorize all the dyes used at an initial dye concentration of 20 mg L⁻¹ at a significantly higher rate as compared to that achieved by individual isolates.     

Kinetic characteristics of bacterial azo-dye decolorization by Pseudomonas luteola

A Pseudomonas luteola strain expressing azoreductase activity was utilized to remove the color of an azo dye (reactive red 22) from contaminated solutions. The effects of substrate concentrations, medium compositions, and operation parameters (e.g., pH, temperature, dissolved oxygen, etc.) on decolorization of the azo dye by a P. luteola strain were systematically investigated to reveal the key factors that dominate the performance of azo-dye decolorization. The metabolites resulting from bacterial decolorization were analyzed by high-performance liquid chromatography (HPLC) and mass spectrometery (MS). The results show that the dissolved oxygen and glucose concentration retarded decolorization of reactive red 22 by P. luteola. The optimal azo-dye decolorization occurred at 37 degrees C, while more rapid decolorization took place over pH 7-9. Yeast extract and tryptone strongly enhanced the decolorization. The Michaelis-Menten model can satisfactorily describe the dependence of specific decolorization rate on the concentration of substrate (reactive red 22 or yeast extract). Decolorization of the azo dye by intact cells of P. luteola was essentially independent of the growth phase, whereas the azoreductase activity of the cell-free extract decreased in the order of late-stationary phase > early-stationary phase > mid-log phase. This suggests that mass transfer of the azo dye across the cell membrane may be the rate-limiting step. The HPLC and MS analyses suggest that both partial reduction and complete cleavage of the azo bond could contribute to decolorization of reactive red 22 by P. luteola.     

Decolorization of an azo dye by unacclimated activated sludge under anaerobic conditions

Studies on the reductive decolorization of a complex azo dye, Reactive Red 3.1, were made as part of the development of a practical approach to better exploit the metabolic potential of biomass in wastewater treatment. Decolorization was achieved at low and variable rates by mixed microbial cultures under various environmental conditions, including low pH and high salt concentration. It was caused by reductive cleavage of the azo bond to yield two aromatic amines. More reliable and effective decolorization rates, of up to 20–30 mg l⁻¹ h⁻¹, were given by unadapted activated sludge, (6 g l⁻¹) incubated with 400 mg l⁻¹ of Reactive Red 3.1 under anaerobic conditions. Decolorization also occurred best in static conditions.     

Decolorization of textile dyes by laccases from a newly isolated strain of Trametes modesta

Four ligninolytic fungi, Trametes modesta, Trametes hirsuta, Trametes versicolor and Sclerotium rolfsii, were compared for their ability to produce laccases. The fungal laccases were screened for their ability to decolorize eight synthetic dyes (anthraquinone, azo, indigo and triarylmethane). The decolorization rate depended both on the source of the enzyme preparation and on the structure of the dye. Based on laccase production and dye decolorizing ability, T. modesta was selected for further studies. All the tested dyes were decolorized by the T. modesta laccase most efficiently under acid conditions (pH 3-6) but the optimum pH for decolorization of the individual dye varied. The decolorization rate of this laccase increased with the rise in temperature to 50-60 degrees C. The decolorization efficiency of T. modesta laccase was improved remarkably in the presence of mediators like 1-hydroxybenzotriazole and 2-methoxyphenothiazine.     

Decolorization and toxicity of reactive anthraquinone textile dyes under methanogenic conditions

Reductive decolorization of two anthraquinone reactive dyes (Reactive Blue 4, RB4; Reactive Blue 19, RB19) under methanogenic conditions was performed using a mixed, methanogenic culture. Decolorization of the two anthraquinone dyes was investigated to evaluate the rate and extent of color removal as well as to assess possible toxic effects of the dyes and their decolorization product(s) on the methanogenic culture as a function of initial dye concentration ranging from 50 to 300 mg x L(-1). A dextrin/peptone mixture was used as the carbon and electron source. A high rate and extent of color removal was achieved ranging from 4.3 to 29.9 mg x L(-1)h(-1) and 73-91% for RB4, and 13.0-74.4 mg x L(-1)h(-1) and 90-95% for RB19. Initial RB4 concentrations up to 100 mg x L(-1) did not result in any significant inhibition. Both the 200 and 300 mg x L(-1) RB4-amended cultures, and all RB19-amended cultures resulted in severe inhibition of both acidogenesis and methanogenesis. Sequential dye addition at 300 mg x L(-1) for both RB4 and RB19 resulted in accumulation of volatile fatty acids (VFAs) and a very low methane production at the end of the first dye addition after 44 days of incubation. However, at the end of the second dye addition, after a relatively long incubation (384 days), recovery of methanogens in the RB4-amended culture was observed in contrast to the complete inhibition of methanogenesis in the RB19-amended culture. Therefore, RB19 resulted in a higher degree of inhibition of both acidogenesis and methanogenesis than RB4. Addition of dextrin/peptone to dye-inhibited cultures resulted in acidogenesis and a gradual recovery of methanogenesis (mainly aceticlastic methanogenesis) in the RB4-inhibited culture, and a slow recovery of acidogenesis but no recovery of methanogenesis in the RB19-inhibited culture. In contrast, addition of 80% H(2)-20% CO(2) gas to dye-inhibited cultures resulted in recovery of hydrogenotrophic methanogenesis in both the RB4- and RB19-inhibited cultures. In spite of the relatively severe inhibition of the two anthraquinone dyes on the mixed, methanogenic culture, a high extent of color removal was achieved.     

Degradation of a textile reactive Azo dye by a combined chemical-biological process: Fenton's reagent-yeast

This work presents the results of our studies on the decolorization of aqueous azo dye Reactive black 5 (RB5) solution combining an advanced oxidation process (Fenton's reagent) followed by an aerobic biological process (mediated by the yeast Candida oleophila). Under our conditions, initial experiments showed that Fenton's process alone, as well as aerobic treatment by C. oleophila alone, exhibited the capacity to significantly decolorize azo dye solutions up to 200 mg/L, within about 1 and 24h, respectively. By contrast, neither Fenton's reagent nor C. oleophila sole treatments showed acceptable decolorizing abilities for higher initial dye concentrations (300 and 500 mg/L). However, it was verified that Fenton's reagent process lowered these higher azo dye concentrations to a value less than 230 mg/L, which is apparently compatible with the yeast action. Therefore, to decolorize higher concentrations of RB5 and to reduce process costs the combination between the two processes was evaluated. The final decolorization obtained with Fenton's reagent process as primary treatment, at 1.0 x 10(-3)mol/L H(2)O(2) and 1.0 x 10(-4)mol/L Fe(2+), and growing yeast cells as a secondary treatment, achieves a color removal of about 91% for an initial RB5 concentration of 500 mg/L.     

Biocalalyst effects of immobilized anthraquinone on the anaerobic reduction of azo dyes by the salt-tolerant bacteria

The accelerating effect of dissolved redox mediators has been studied in details in the bio-decolorization processes, but there are little literatures about the non-dissolved redox mediators. Here we describe the accelerating effect of anthraquinone as a redox mediator in the bio-decolorization. Decolorization of azo dyes was carried out experimentally using the salt-tolerant bacteria under immobilized anthraquinone and high salt conditions. Anthraquinone as a redox mediator was able to increase the decolorization rate of azo dyes, and was immobilized by entrapment in calcium alginate (CA), Polyvinyl alcohol (PVA)-H(3)BO(3) and agar, respectively. The effects of various operating conditions such as anthraquinone bead number, dissolved oxygen, temperature and pH on microbial decolorization were investigated experimentally. The reusability of the anthraquinone immobilization beads was evaluated with repeated-batch decolorization experiments. After four repeated experiments, the decolorization rate of CA immobilized anthraquinone retained over 90% of their original value. The experiments explored a great improvement of the redox mediator application and the new bio-treatment concept.     

Optimisation of process parameters for bioreduction of azo dyes using Bacillus firmus under batch anaerobic condition

A new dye decolourising bacterial strain was isolated from textile wastewater and identified as Bacillus firmus. The study indicated that the bacterium could efficiently decolourise different azo dyes under static culture conditions. Characterisation of the efficiency of azo dye reduction by this isolate using both spectral and HPLC analysis was found to be a function of process parameters which include dye concentration, culture broth pH, incubation temperature, aeration as well as nitrogen source. For decolourisation, the optimal pH and temperature were 7–8 and 20–35°C respectively, while remarkable dye degradation was obtained within 18 h for dye concentrations below 100 mg L^?1. With the addition of yeast extract and under optimal conditions, dye reduction was enhanced and complete colour removal was achieved within 12 h. Colour removal was shown to be due to biodegradation rather than adsorption of dyes on bacterial cells. This study confirms the ability of the new dye‐degrading strain, Bacillus firmus, to decolourise and degrade different azo dyes and highlights its high biotechnology potential for the eco‐friendly treatment of textile wastewater when optimal conditions are applied.     

Enzymatic biodegradation and detoxification of azo and anthraquinone dyes by indigenously isolated bacterial monocultures and their synergistic behaviour in developed consortia

Wastewater generated by textile industry needs to be treated to reduce its toxicity before final disposal and/or for recycling purposes. In the current study, several bacterial strains were screened for dye decolorization potential. UV–visible spectroscopy was used to determine maximum absorption wavelength of disperse dyes. HPLC and MTS assay were used to confirm the degradation and detoxification of disperse dyes, respectively. Results revealed that indigenously isolated Bacillus licheniformis, Glutamicibacter uratoxydans and Pseudomonas aeruginosa showed strong decolorization of red, blue and violet, respectively in 6–9 h. MTS assay revealed 100% viability of NIH/3T3 cell lines in presence of treated dyes. Enzyme screening assay confirmed the production of intracellular and membrane bound oxidoreductases in presence of specific dye as substrate. To resolve this issue, bacterial consortia were prepared, and better decolorization of all dyes was achieved in synergistic behaviour of Consortia 1 and 4 with 85% and 88% decolorization potential, respectively.     

Metabolism of azo dyes by Lactobacillus casei TISTR 1500 and effects of various factors on decolorization

Lactobacillus casei TISTR 1500 was isolated from soil of a dairy wastewater treatment plant and selected as the most active azo dye degrader of 19 isolates. Growing cells and freely suspended cells of this strain completely degraded methyl orange, thereby decolorizing the medium. The strain stoichiometrically converted methyl orange to N,N-dimethyl-p-phenylenediamine and 4-aminobenzenesulfonic acid, which were identified by HPLC, GC, and GC-MS analyses. The enzyme activity responsible for the cleavage of the azo bond of methyl orange was localized to the cytoplasm of cells grown on modified MRS medium containing methyl orange. The effect of sugars, oligosaccharides, organic acids, metal ions, pHs, oxygen and temperatures on methyl orange decolorization by freely suspended cells was investigated. The optimal conditions for the decolorization of methyl orange by the Lactobacillus casei TISTR 1500 are incubation at 35 degrees C and pH 6 with sucrose provided as the energy source.     

Laccase-membrane reactors for decolorization of an acid azo dye in aqueous phase: Process optimization

In the present investigation, performance of various laccase-membrane reactor configurations including direct enzyme contact, enzyme impregnated, immobilized enzyme and a reactor system based on laccase immobilization in chitosan membranes for decolorization of azo dye (acid black 10 BX) were examined using laccase enzyme purified from white rot fungi Pleurotus ostreatus 1804. A five-step laccase purification procedure was employed, which improved the enzymatic activity by 8.27 folds. Laccase was confirmed by comparing with the standard marker using SDS-PAGE electrophoresis, which showed molecular weight of 63 kDa. Experimental data showed that laccase has great potential for color removal without addition of external redox mediators. Various process parameters viz. aqueous phase of pH 6.0, enzyme concentration of 1.75 U/ml, dye concentration of 20 mg/L, temperature of 30 °C and reaction time of 120 min were optimized to achieve maximum decolorization efficiencies. Moreover, different laccase-membrane reactor configurations were tested to determine the efficacy of repeated application of laccase on dye decolorization process. Among the different reactor configurations employed, laccase encapsulated in chitosan membrane showed advantages such as short-term contact period and reusability of enzyme for a number of cycles.     

Application of the laccase,produced on coconut flesh by Pleurotus florida for dye decolorization

We investigated the ability of Pleurotus florida to produce laccase on coconut flesh as a solid substrate fermentation. The decolorization of two structurally different dyes such as azo (Reactive Blue 198) and triphenylmethane dye (Malachite Green) were analysed. The decolorization of Reactive blue 198 and Malachite Green at 8 hrs was 93% and 63% respectiely. The untreated and treated dye was characterized by UV-Vis spectral and fourier transform infrared (FTIR) Spectroscopy scan. FTIR analysis pointed out the involvement of alkene (C=C) and carboxylic (C-O) groups in the decolorization process. The toxicity with respect to Allium cepa root inhibition was measured to demonstrate the potential of laccase in the detoxication and bioremediation process.     

High performance degradation of azo dye Acid Orange 7 and sulfanilic acid in a laboratory scale reactor after seeding with cultured bacterial strains

Bacterial strains 1CX and SAD4i--previously isolated from the mixed liquor of a municipal sewage treatment plant--are capable of degrading the azo dye Acid Orange 7 (AO7) and sulfanilic acid, respectively. A rotating drum bioreactor (RDBR), operating under continuous flow and nutrient conditions designed to simulate the effluent from a dye manufacturing plant, was seeded with strains 1CX and SAD4i, forming a biofilm capable of degrading AO7 and sulfanilic acid. In addition, an RDBR containing a pre-existing biofilm capable of degrading AO7, but not sulfanilic acid, was seeded with strain SAD4i alone. Strain SAD4i was incorporated into the existing biofilm and degraded the sulfanilic acid resulting from the degradation of AO7 by indigenous members of the biofilm. The ability to seed a bioreactor with bacterial strains capable of degrading azo dyes, and resulting by-products, in a mixed microbial community suggests that this process could have commercial applications.     

Toxicological effect of indole and its azo dye derivatives on some microorganisms under aerobic conditions

Azo dyes are ubiquitous commercial chemicals that present unique environmental problems. The azo dyes in particular can undergo natural anaerobic degradation to potentially carcinogenic amines. They pose a major problem for water-treatment plants downstream. One strategy to remidate polluted surface contaminants is to make use of the degradative capacity of bacteria rather than using destructive chemical reactions. Therefore, pathogenic and non-pathogenic microorganisms namely; Bacillus thuringiensis, Bacillus subtilis, Bacillus megaterium, Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Saccharomyces cerevisiae have been chosen and the toxicological effect of indole and its azo dye methyl derivatives on these microorganisms was studied under aerobic conditions. While these compounds have showed remarkable activity against B. megaterium, B. subtilis, B. thuringiensis and S. aureus, they did not exhibit any activity against P. vulgaris. However, indole acted as an inhibitor on all of these compounds specially the Gram negative bacterium P. aeruginosa.     

Application of 'waste' metal hydroxide sludge for adsorption of azo reactive dyes

The capacity and mechanism of metal hydroxide sludge in removing azo reactive dyes from aqueous solution was investigated with different parameters, such as charge amount of dyes, system pH, adsorbent particle size, and adsorbent dosage. The three anionic dyes used were CI Reactive Red 2, CI Reactive Red 120, and CI Reactive Red 141, increasing in number of sulfonic groups, respectively. Only 0.2% (w/v) of powdered sludge (<75microm) achieved color removal from 30 mg l(-1) reactive dye solutions within 5 min without pH adjustment. The larger the charge amount of the dyes, the greater the adsorption (>90%) on the metal hydroxide sludge. The system pH played a significant role in the adsorption on metal hydroxides and formation of dye-metal complexes. The optimum system pH for dye adsorption was 8-9 which was close to the pH(zpc) of the sludge while the precipitation of dye-metal complexes occurred at system pH 2. The maximum adsorption capacity (Q degrees ) of the sludge for the reactive dyes was 48-62 mg dye g(-1) adsorbent. The Langmuir and Freundlich models showed that the higher charged dyes had a higher affinity of adsorption. The smaller particle size and the greater amount of adsorbent showed the faster process, due to an increase in surface area of adsorbent. Desorption studies elucidated that metal hydroxide sludge had a tendency for ion exchange adsorption of sulfonated azo reactive dyes. Leaching data showed that the treated water was nontoxic at a system pH above 5 or a solution pH above 2.     

Bioaugmented membrane bioreactor (MBR) with a GAC-packed zone for high rate textile wastewater treatment

The long-term performance of a bioaugmented membrane bioreactor (MBR) containing a GAC-packed anaerobic zone for treatment of textile wastewater containing structurally different azo dyes was observed. A unique feeding strategy, consistent with the mode of evolution of separate waste streams in textile plants, was adopted to make the best use of the GAC-zone for dye removal. Dye was introduced through the GAC-zone while the rest of the colorless media was simultaneously fed through the aerobic zone. Preliminary experiments confirmed the importance of coupling the GAC-amended anaerobic zone to the aerobic MBR and also evidenced the efficacy of the adopted feeding strategy. Following this, the robustness of the process under gradually increasing dye-loading was tested. The respective average dye concentrations (mg/L) in the sample from GAC-zone and the membrane-permeate under dye-loadings of 0.1 and 1 g/L.d were as follows: GAC-zone (3, 105), permeate (0, 5). TOC concentration in membrane-permeate for the aforementioned loadings were 3 and 54 mg/L, respectively. Stable decoloration along with significant TOC removal during a period of over 7 months under extremely high dye-loadings demonstrated the superiority of the proposed hybrid process.     

Bioremediation of crystal violet using air bubble bioreactor packed with Pseudomonas aeruginosa

Seven water and sediment samples were collected and tested for decolorizing crystal violet. Pseudomonas aeruginosa was the most effective isolate for dye decolorization. The LC₅₀ of the crystal violet (115 mg/l) was measured using Artemia salina as a biomarker. The effect of different heavy metals on crystal violet decolorization was investigated. Cd²⁺ and Fe³⁺ ions showed marginal enhancement of the decolorization process, the rate was 1.35 mg/l/h compared to (1.25 mg/l/h) for the control. Phenol and m-cresol showed no effect on crystal violet decolorization, meanwhile p-cresol and p-nitrophenol reduced the decolorization rate to 1.07 and 0.01 mg/l/h, respectively. P. aeruginosa cells were immobilized by entrapment in agar-alginate beads. The beads were cultivated and reused in Erlenmeyer flask and in an air bubble column bioreactor and they enhanced the crystal violet decolorization rate to 3.33 and 7.5 mg/l/h, respectively.     

Advanced oxidation of a reactive dyebath effluent: comparison of O3, H2O2/UV-C and TiO2/UV-A processes

In the present study the treatment efficiency of different AOPs (O3/OH- H2O2/UV-C and TiO2/UV-A) were compared for the oxidation of simulated reactive dyebath effluent containing a mixture of monochlorotriazine type reactive dyes and various dye auxiliary chemicals at typical concentrations encountered in exhausted reactive dyebath liquors. A525 (color), UV280 (aromaticity) and TOC removal rates were assessed to screen the most appropriate oxidative process in terms of reactive dyebath effluent treatment. Special emphasis was laid on the effect of reaction pH and applied oxidant (O3, H2O2) dose on the observed reaction kinetics. It was established that the investigated AOPs were negatively affected by the Na2CO3 content (= 867 mg/L) which is always present at high concentrations in dychouse effluents since it is applied as a pH buffer and dye fixation agent during the reactive dyeing process. The ozonation reaction exhibited almost instantaneous decolorization kinetics and a reasonable TOC reduction rate. It appeared to be stable under the investigated advanced oxidation conditions and outranked the other studied AOPs based on the above mentioned criteria. Besides, the electrical energy requirements based on the EE/O parameter (the electrical energy required per order of pollutant removal in 1 m3 wastewater) was calculated for the homogenous AOPs in terms of decolorization kinetics. In view of the electrical energy efficiency, ozonation and H2O2/UV-C oxidation at the selected treatment conditions appear to be promising candidates for full-scale dyehouse effluent decolorization.     

Bioremediation of crystal violet using air bubble bioreactor packed with Pseudomonas aeruginosa

Seven water and sediment samples were collected and tested for decolorizing crystal violet. Pseudomonas aeruginosa was the most effective isolate for dye decolorization. The LC₅₀ of the crystal violet (115 mg/l) was measured using Artemia salina as a biomarker. The effect of different heavy metals on crystal violet decolorization was investigated. Cd²⁺ and Fe³⁺ ions showed marginal enhancement of the decolorization process, the rate was 1.35 mg/l/h compared to 1.25 mg/l/h for the control. Phenol and m-cresol showed no effect on crystal violet decolorization, meanwhile p-cresol and p-nitrophenol reduced the decolorization rate to 1.07 and 0.01 mg/l/h, respectively. P. aeruginosa cells were immobilized by entrapment in agar–alginate beads. The beads were cultivated and reused in Erlenmeyer flask and in an air bubble column bioreactor and they enhanced the crystal violet decolorization rate to 3.33 and 7.5 mg/l/h, respectively.