Azo dye decolorization with a mutant Escherichia coli strain

A recombinant Escherichia coli strain (E. coli NO3) containing genomic DNA fragments from azo-reducing wild-type Pseudomonas luteola strain decolorized a reactive azo dye (C.I. Reactive Red 22) at approx. 17 mg dye h^–1 g cell. The ability to decolorize the azo dye probably did not originate from the plasmid DNA. Acclimation in azo-dye-containing media gave a nearly 10% increase in the decolorization rate of E. coli NO3. Growth with 1.25 g glucose l^–1 completely stopped the decolorization activity. When the decolorization metabolites from E. coli NO3 were analyzed by HPLC and MS, the results suggested that decolorization of the azo dye may be due to cleavage of the azo bond.     

Degradation of dye-containing textile effluent by the agaric white-rot fungus Clitocybula dusenii

Raw mixed-dye wastewater from a textile dye-producing plant was partly decolorized by the agaric white-rot fungus, Clitocybula dusenii. The fungus had higher Mn peroxidase (MnP) and laccase activities when grown with dye effluent than in control cultures. The activity of MnP increased commensurately with the proportion of the raw dye wastewater in the medium (control: 20 U l^–1; 10% v/v effluent: 67 U l^–1; 25% v/v effluent: 130 U l^–1; and 33% v/v effluent: 180 U l^–1). Maximal decolorization rates were achieved over 20 d at 28 °C using four-fold diluted dye-containing effluent on a 5 d pre-grown mycelium.     

Decolorization kinetics of a recombinant Escherichia coli strain harboring azo-dye-decolorizing determinants from Rhodococcus sp.

A 6.3 kb DNA fragment containing genes responsible for azo-dye decolorization was cloned and expressed in Escherichia coli. The resulting recombinant strain E. coli CY1 decolorized 200 mg azo dye (C.I. Reactive Red 22) l^–1 at 28 °C at 8.2 mg g cell^–1 h^–1, while the host (E. coli DH5) had no color-removal activity. Addition of 0.5 mM isopropyl--d-thiogalacto-pyranoside (IPTG) increased the decolorization rate 3.4-fold. The dependence of the decolorization rate on initial dye concentration essentially followed Monod-type kinetics and the maximal rate occurred with the dye at 600 mg l^–1. The decolorization rate of E. coli CY1 was optimal at 40 °C and pH 11. Aeration (increased dissolved O₂ level) strongly inhibited the decolorization, but decolorization occurred effectively under static incubation conditions (no agitation was employed). The CY1 strain also exhibited excellent stability during repeated-batch operations.     

Amelioration of ligninolytic enzyme production by Phanerochaete chrysosporium in airlift bioreactors

Maximum activities of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) in free cultures of Phanerochaete chrysosporium (ATCC 24725) were 258 U l^–1 and 103 U l^–1, respectively, in an airlift bioreactor. Immobilisation of the fungus on an inert carrier as well as several design modifications of the bioreactor employed gave MnP activities around 500–600 U l^–1 during 9 days' operation. The continuous operation of the latter led to MnP and LiP activities about 140 U l^–1 and 100 U l^–1, respectively, for two months, without operational problems. Furthermore, the extracellular liquid secreted decolourised the polymeric dye Poly R-478 about 56%.     

Decolourization of synthetic dyes by a newly isolated strain of Serratia marcescens

A novel dye-decolourizing strain of the bacterium Serratia marcescens efficiently decolourized two chemically different dyes Ranocid Fast Blue (RFB) and Procion Brilliant Blue-H-GR (PBB-HGR) belonging respectively to the azo and anthraquinone groups. Extracellular laccase and manganese peroxidase (MnP) activity were detected during dye decolourization. The involvement of MnP activity was found in the decolourization of both dyes. More than 90% decolourization of PBB-HGR and RFB was obtained on days 8 and 5, respectively at 26 °C under static conditions at pH 7.0. MnP activity was increased by the addition of Mn²⁺ · At 50 M Mn²⁺, high MnP (55.3 U/ml) but low laccase activity (8.3 U/ml) was observed. Influence of oxalic acid on MnP activity was also observed.     

Decolorization Screening of Synthetic Dyes by Anaerobic Methanogenic Sludge Using a Batch Decolorization Assay

The nonspecific ability of anaerobic sludge bacteria obtained from cattle dung slurry was investigated for 17 different dyes in a batch assay system using sealed serum vials. Experiments using Reactive Violet 5 (RV 5) showed that sludge bacteria could effectively decolorize solutions having dye concentrations up to 1000 mg l⁻¹ with a decolorization efficiency of above 75% during 48 h of incubation. Headspace gas composition of anaerobic batch systems for varying dye concentration revealed that lower concentrations of RV 5 (upto 500 mg l⁻¹) were found to be stimulatory to the methanogenic activity of sludge bacteria. However at higher dye concentrations, the headspace gas composition was found to be similar to batch assay controls without dye, indicating that dye at higher concentrations was inhibitory to methanogenic bacteria of sludge. The optimum inoculum and incubation temperature for maximum decolorization of RV 5 was found to be 9.0 g l⁻¹(in terms of total solids) and 37°C, respectively. Of sixteen other dyes tested, nine (Reactive Black 5, Reactive Blue 31, Reactive Blue 28, Reactive Red HE8B, Reactive Yellow, Reactive Golden Yellow, Mordant Orange, Novatic Olive R S/D & Navilan Yellow GL) were decolorized with more than 88% efficiency; three (Orange II, Navy Blue HER & Novatic Blue BC S/D) were decolorized with about 50–65% efficiency, whereas other three dyes (Procion Orange H2R, Procion Brilliant Blue HGR & Novatic Blue BC S/D) were decolorized with less than 40% efficiency. Though Ranocid Fast Blue was decolorized with about 92.5% efficiency, this was merely due to sorption, whereas the other dyes were decolorized due to biotransformation.     

Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor

The production of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) by the fungus Phanerochaete chrysosporium (ATCC 24725) in a new bioreactor, the Immersion Bioreactor, which grows cells under solid-state conditions, was studied. Maximum MnP and LiP activities were 987 U l^–1 and 356 U l^–1, respectively. The polymeric dye, Poly R-478, was degraded at 2.4 mg l^–1 min^–1 using the extracellular culture filtrate.     

Decolorization kinetics of the azo dye Reactive Red 2 under methanogenic conditions: effect of long-term culture acclimation

The biological decolorization of the textile azo dye Reactive Red 2 was investigated using a mixed, mesophilic methanogenic culture, which was developed with mixed liquor obtained from a mesophilic, municipal anaerobic digester and enriched by feeding a mixture of dextrin/peptone as well as media containing salts, trace metals and vitamins. Batch decolorization assays were conducted with the unacclimated methanogenic culture and dye decolorization kinetics were determined as a function of initial dye, biomass, and carbon source concentrations. Dye decolorization was inhibited at initial dye concentrations higher than 100 mg l^-1 and decolorization kinetics were described based on the Haldane model. The effect of long-term culture exposure to the reactive dye on decolorization kinetics, culture acclimation, as well as possible dye mineralization was tested using two reactors fed weekly for two years with an initial dye concentration of 300 mg l^-1 and a mixture of dextrin/peptone. The maximum dye decolorization rate after a 2-year acclimation at an initial dye concentration of 300 mg l^-1 was more than 10-fold higher as compared to that obtained with the unacclimated culture. Aniline and the o-aminohydroxynaphthalene derivative resulting from the reductive azo bond cleavage of the dye were detected, but further transformation(s) leading to dye mineralization were not observed. Reactive Red 2 did not serve as the carbon and energy source for the mixed culture, and dye decolorization was sustained by the continuous addition of dextrin and peptone. Thus, biological decolorization of reactive azo dyes is feasible under conditions of low redox potential created and maintained in overall methanogenic systems, but supply of a biodegradable carbon source is necessary.     

Biodecolorization of textile azo dyes by isolated yeast from activated sludge: Issatchenkia orientalis JKS6

An ascomycetous yeast strain isolated from activated sludge could decolorize Reactive Black 5 azo dye at 200 mg l^?1 up to 90 % within 12–18 h under agitated condition. Yeast decolorization ability was investigated at different RB5 concentrations and, at higher dye concentration, 500 mg l^?1, the decolorization was found to be 98 % after 36 h incubation time. Extensive decolorization (95–99 %) was obtained in presence of five other azo dyes, Reactive Orange 16, Reactive Red 198, Direct Blue 71, Direct Yellow 12, and Direct Black 22, by isolated yeast. HPLC analysis, UV–vis spectra and colorless biomass obtained after complete decolorization showed that the decolorization occured through a biodegradation mechanism. Decolorization was occurred during the exponential growth phase which is associated to primary metabolism. Laccase production by the yeast cells was not detected. The isolated yeast was characterized according to phenotypical and molecular procedures and was closely related (99 % identity) to Issatchenkia orientalis.     

Effect of various synthetic dyes on the production of manganese-dependent peroxidase isoenzymes by immobilized <Emphasis Type="Italic">Irpex lacteus</Emphasis>

The effect of manganese and selected synthetic dyes on the production of manganese-dependent peroxidase (MnP) by Irpex lacteus immobilized on polyurethane foam was studied. In the cultures grown in a medium containing 65 μM Mn (II), up to three various isoenzymes of MnP were resolved by isolectrofocusing, with pI values within the range of 3.50–6.04. In the cultures grown in a medium containing 2.9 mM Mn (II), two new MnP isoforms (pI 3.28, 3.75) were produced. The addition of structurally different synthetic dyes, an azo dye Reactive Orange 16 (RO16), an anthraquinonic dye Remazol Brilliant Blue R (RBBR), and a triphenylmethane dye Bromophenol Blue (BPB), to the fungal cultures grown in the presence of high manganese inhibited the production of low pI MnP isoforms. However, in the presence of BPB a new MnP isoform with pI 5.67 was detected. BPB was found to induce MnP isoforms which are more effective in RBBR decolorization in vitro than the low pI isoforms present in the control cultures.     

Optimization of manganese peroxidase and laccase production in the South American fungus<Emphasis Type="Italic"> Fomes sclerodermeus</Emphasis> (Lév.) Cke

Fomes sclerodermeus produces manganese peroxidase (MnP) and laccase as part of its ligninolytic system. A Doehlert experimental design was applied in order to find the optimum conditions for MnP and laccase production. The factors studied were Cu²⁺, Mn²⁺ and asparagine. The present model and data analysis allowed us not only to define optimal media for production of both laccase and MnP, but also to show the combined effects between the factors. MnP was strongly influenced by Mn²⁺, which acts as an inducer. Under these conditions Cu²⁺ negatively affected MnP activity. At 13 days of growth 0.75 U ml^–1 were produced in the optimized culture medium supplemented with 1 mM MnSO₄ and 4 g l^–1 asparagine. The laccase titer under optimized conditions reached maximum values at 16 days of growth: 13.5 U ml^–1 in the presence of 0.2 mM CuSO₄, 0.4 mM MnSO₄ and 6 g l^–1 asparagine. Mn²⁺ promoted production of both enzymes. There were important interactions among the nutrients evaluated, the most significant being those between Cu²⁺ and asparagine.     

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.     

Decolorization of textile dyestuffs by a mixed bacterial consortium

A mixed anaerobic bacterial culture decolorized Drimaren Orange K-GL, Everzol Red RBN and Everdirect Supra Yellow PG dyestuffs at 200 mg dyestuff l^–1 over 24 h. Improved performance with complete decolorization within 24 h was achieved by incubation with 5 g yeast extract l^–1 compared to glucose, lactose and sucrose though 50 mg yeast extract l^–1 supplemented with 5 g lactose l^–1 or 5 g sucrose l^–1 also resulted in complete decolorization within 24 h.     

Dynamics of microbial community for X-3B wastewater decolorization coping with high-salt and metal ions conditions

In this study, decolorization of Reactive Brilliant Red X-3B wastewater by the biological process coping with high salinity and metal ions conditions was investigated, and 16S rDNA based fingerprint technique was used to investigate microbial population dynamics. Results of sequencing batch tests showed that the microbial community could keep efficient with high concentration of dye (1100 mg L⁻¹), salt (150 g L⁻¹ NaCl) and some metal ions such as Mg²⁺, Ca²⁺ (1–10 mmol L⁻¹) and Pb²⁺ (1 mmol L⁻¹). 16S rDNA-based molecular analysis techniques demonstrated that the microbial community shifted during the acclimatization process affected by salt or metal ions. Some stains similar to Bacillus, Sedimentibacter, Pseudomonas, Clostridiales, Streptomyces and some uncultured clones acted for the dynamic succession, supposed as potential decolorization bacteria. This study provided insights on the decolorization capability and the population dynamic shifts during decolorization process of azo dye wastewater coping with salt and metal ions.     

Biodegradation of Methyl red by Galactomyces geotrichum MTCC 1360

Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane, azo and reactive high exhaust textile dyes. At shaking condition this strain showed 100% decolorization of a toxic azo dye Methyl red (100 m gl⁻¹) within 1 h in deionized water at 30 °C. The degradation of Methyl red was possible through a broad pH (3–12) and temperature (5–50 °C) range. Glucose and mycelium concentration had increased the decolorization rate, but the addition of 1 gl⁻¹ molasses in deionized water made decolorization possible in only 10 min. Induction in the NADH–dichloro phenol indophenol (NADH–DCIP) reductase, Malachite green reductase, laccase and lignin peroxidase (Lip) activities were observed in the cells obtained after complete decolorization, showing that there is direct involvement in the degradation of Methyl red. The absence of N-N′-dimethyl-p-phenylenediamine (DMPD) in 5 °C, 2-aminobenzoic acid (ABA) in 50 °C and both the compounds in 30 °C sample have shown the differences in the metabolic fate of Methyl red at different temperatures. The untreated dye at 300 mg l⁻¹ concentration showed 88% germination inhibition in Sorghum bicolor, whereas it was 72% in Triticum aestivum. There was no germination inhibition for both the plants by Methyl red metabolites at 300 mg l⁻¹ concentration.

The scientific relevance of the paper

The azo dye Methyl red (100 mg l⁻¹) was decolorized by G. geotrichum MTCC 1360 within 1 h at shaking condition in deionized water. This organism could decolorize Methyl red at wide pH and temperature ranges. Decolorization time was reduced to 10 min by the addition of molasses to deionized water. There was induction in laccase and Lip, NADH–DCIP reductase and Malachite green reductase activities. The metabolic fate of Methyl red changes with temperature which can be evidenced by the formation of 2-ABA at 5 °C, N-N′-DMPD at 50 °C and both the compounds were absent at 30 °C. Phytotoxicity showed that metabolites of dye had induced shoot and root length of both the tested plants.     

Decolorization of azo dyes by Rhodobacter sphaeroides

Rhodobacter sphaeroides AS1.1737 decolorized more than 90% of several azo dyes (200 mg dyes l^–1) in 24 h. The optimal culture conditions were: anaerobic illumination (1990 lx), peptone as carbon source, temperature 35–40 °C and pH 7–8. Intracellular crude enzyme from this strain had azoreductase activity, optimized temperature as 45–50 °C, and decolorization kinetics which were consistent with a ping-pong mechanism.     

Transformation of Industrial Dyes by Manganese Peroxidases from Bjerkandera adusta and Pleurotus eryngii in a Manganese-Independent Reaction

We investigated the transformation of six industrial azo and phthalocyanine dyes by ligninolytic peroxidases from Bjerkandera adusta and other white rot fungi. The dyes were not oxidized or were oxidized very little by Phanerochaete chrysosporium manganese peroxidase (MnP) or by a chemically generated Mn³⁺-lactate complex. Lignin peroxidase (LiP) from B. adusta also showed low activity with most of the dyes, but the specific activities increased 8- to 100-fold when veratryl alcohol was included in the reaction mixture, reaching levels of 3.9 to 9.6 U/mg. The B. adusta and Pleurotus eryngii MnP isoenzymes are unusual because of their ability to oxidize aromatic compounds like 2,6-dimethoxyphenol and veratryl alcohol in the absence of Mn²⁺. These MnP isoenzymes also decolorized the azo dyes and the phthalocyanine complexes in an Mn²⁺-independent manner. The reactions with the dyes were characterized by apparent K_m values ranging from 4 to 16 μM and specific activities ranging from 3.2 to 10.9 U/mg. Dye oxidation by these peroxidases was not increased by adding veratryl alcohol as it was in LiP reactions. Moreover, the reaction was inhibited by the presence of Mn²⁺, which in the case of Reactive Black 5, an azo dye which is not oxidized by the Mn³⁺-lactate complex, was found to act as a noncompetitive inhibitor of dye oxidation by B. adusta MnP1.     

Decolorization of azo dye by immobilized Pseudomonas luteola entrapped in alginate–silicate sol–gel beads

Pseudomonas luteola was immobilized by entrapment in alginate–silicate sol–gel beads for decolorization of the azo dye, Reactive Red 22. The influences of biomass loading and operating conditions on specific decolorization rate and dye removal efficiency were studied in details. The immobilized cells were found to be less sensitive to changes in agitation rates (dissolved oxygen levels) and pH values. Michaelis–Menten kinetics could be used to describe the decolorization kinetics with the kinetic parameters being 36.5 mg g⁻¹ h⁻¹, 300.1 mg l⁻¹ and 18.2 mg g⁻¹ h⁻¹, 449.8 mg l⁻¹ for free and immobilized cells, respectively. After five repeated batch cycles, the decolorization rate of the free cells decreased by nearly 54%, while immobilized cells still retained 82% of their original activity. The immobilized cells exhibited better thermal stability during storage and reaction when compared with free cells. From SEM observation, a dense silicate gel layer was found to surround the macroporous alginate–silicate core, which resulted in much improved mechanical stability over that of alginate beads when tested under shaking conditions. Alginate–silicate matrices appeared to be the best matrix for immobilization of P. luteola in decolorization of Reactive Red 22 when compared with previous results using synthetic or natural polymer matrices.     

Decolorization of anthraquinone dye by Shewanella decolorationis S12

A new species of genus Shewanella, Shewanella decolorationis S12, from activated sludge of a textile-printing wastewater treatment plant, can decolorize Reactive Brilliant Blue K-GR, one kind of anthraquinone dye, with flocculation first. Although S. decolorationis displayed good growth in an aerobic condition, color removal was the best in an anaerobic condition. For color removal, the most suitable pH values and temperatures were pH 6.0–8.0 and 30–37°C under anaerobic culture. More than 99% of Reactive Brilliant Blue K-GR was removed in color within 15 h at a dye concentration of 50 mg/l. Lactate was the suitable carbon source for the dye decolorization. A metal compound, HgCl₂, had the inhibitory effect on decolorization of Reactive Brilliant Blue K-GR, but a nearly complete decolorization also could be observed at a HgCl₂ concentration of 10 mg/l. The enzyme activities, which mediate the tested dye decolorization, were not significantly affected by preadaptation of the bacterium to the dye.