Degradation of textile dyes mediated by plant peroxidases

The peroxidase enzyme from the plants Ipomea palmata (1.003 IU/g of leaf) and Saccharum spontaneum (3.6 IU/g of leaf) can be used as an alternative to the commercial source of horseradish and soybean peroxidase enzyme for the decolorization of textile dyes, mainly azo dyes. Eight textiles dyes currently used by the industry and seven other dyes were selected for decolorization studies at 25–200 mg/L levels using these plant enzymes. The enzymes were purified prior to use by ammonium sulfate precipitation, and ion exchange and gel permeation chromatographic techniques. Peroxidase of S. spontaneum leaf (specific activity of 0.23 IU/mg) could completely degrade Supranol Green and Procion Green HE-4BD (100%) dyes within 1 h, whereas Direct Blue, Procion Brilliant Blue H-7G and Chrysoidine were degraded >70% in 1 h. Peroxidase of Ipomea (I. palmata leaf; specific activity of 0.827 U/mg) degraded 50 mg/L of the dyes Methyl Orange (26%), Crystal Violet (36%), and Supranol Green (68%) in 2–4 h and Brilliant Green 54%), Direct Blue (15%), and Chrysoidine (44%) at the 25 mg/L level in 1 to 2 h of treatment. The Saccharum peroxidase was immobilized on a hydrophobic matrix. Four textile dyes, Procion Navy Blue HER, Procion Brilliant Blue H-7G, Procion Green HE-4BD, and Supranol Green, at an initial concentration of 50 mg/L were completely degraded within 8 h by the enzyme immobilized on the modified polyethylene matrix. The immobilized enzyme was used in a batch reactor for the degradation of Procion Green HE-4BD and the reusability was studied for 15 cycles, and the halflife was found to be 60 h.     

Improved Biodegradation of Synthetic Azo Dye by Anionic Cross-Linking of Chloroperoxidase on ZnO/SiO<Subscript>2</Subscript> Nanocomposite Support

A novel ZnO nanowire/macroporous SiO₂ composite was used as a support to immobilize chloroperoxidase (CPO) by in situ cross-linking method. An anionic bi-epoxy compound was synthesized and used as a long-chained anionic cross-linker, and it was adsorbed on the surface of ZnO nanowires through static interaction before reaction with CPO, creating a new approach to change the structure, property, and catalytic performance of the produced cross-linking enzyme aggregates (CLEAs) of CPO. The immobilized CPO showed high activity in the decolorization of three azo dyes. The effect of various conditions such as the loading amount of CPO, solution pH, temperature, and dye concentration was optimized on the decolorization. Under optimized conditions, the decolorization percentage of Acid Blue 113, Direct Black 38, and Acid Black 10 BX reached as high as 95.4, 92.3, and 89.1%, respectively. The immobilized CPO exhibited much better thermostability and resistance to pH inactivation than free CPO. The storage stability and reusability were greatly improved through the immobilization. It was found from the decolorization of Acid Blue 113 that 83.6% of initial activity retained after incubation at 4 °C for 60 days and that 80.9% of decolorization efficiency retained after 12 cycles of reuses.     

Identification and Characteristics of A Novel Salt-Tolerant <Emphasis Type="Italic">Exiguobacterium</Emphasis> sp. for Azo Dyes Decolorization

A salt-tolerant bacterium was isolated from the surface soil of a pharmaceutical factory, which could efficiently decolorize azo dyes. The strain was identified as Exiguobacterium sp. according to its morphological characteristics and 16S rRNA gene sequence analysis. Decolorization of X-3B with resting cells of this strain, which were catalyzed by redox mediator (anthraquinone), was studied, and the conditions were optimized. For color removal and cells growth, the optimal inoculation amount, pH, temperature, salinity, and metal ions were 6% (v/v), 5.4–7.0, 30–40 °C, 15% (w/v) NaCl, and 1 mmol L⁻¹ Mg²⁺ or Ca²⁺, respectively. It was exhibited that decolorization process proceeded primarily by enzymatic reduction associated with a minor portion of bio-adsorption to inactivated microbial cells. Anthraquinone could really accelerate the decolorization of X-3B under the optimal conditions.     

Modified <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis> biomass for sorption of <Superscript>241</Superscript>Am and other radionuclides

The improvement and the refinement of non-viable Rhizopus arrhizus biomass were investigated via immobilization. Immobilization was carried out by using sodium alginate/CaCl₂ solution and formaldehyde/HCl cross-linking with dead Rhizopus arrhizus biomass and were used for the sorption of radionuclides from low level effluent wastes. The sodium alginate/CaCl₂ immobilized biomass (ratio 1:2) showed about 86% sorption for ²⁴¹Am activity but due to its soft nature and tendency to undergo distortion in shape, is unsuitable for practical applications. The biomass cross-linked with 15% formaldehyde/0.1 M HCl solution has a relatively high mechanical strength and rigidity. It was showing a sorption of >99% for ²⁴¹Am activity and has the sorption capacity of ~65 mg/g for americium and uranium. Hence, it can be utilized for the removal of radionuclides from radioactive waste effluents.     

Continuous production of butanol with immobilized cells ofClostridium acetobutylicum

Spores ofClostridium acetobutylicum were immobilized in calcium alginate. An active gel preparation was obtained after outgrowth of the spores to vegetative cells within the gel matrix. A 100 mL column containing the immobilized cells was used for continuous production. At steady-state conditions the productivity of butanol was 67 g/L reactor volume/day.     

A comparative study on biosorption characteristics of certain fungi for bromophenol blue dye

Laboratory investigations of the potential use of dried biomasses of Rhizopus stolonifer, Fusarium sp., Geotrichum sp., and Aspergillus fumigatus as biosorbents for the removal of bromophenol blue (BPB) dye from aqueous solutions were conducted. Kinetics studies indicated that the BPB dye uptake processes can be well described by the pseudo-second-order model. The fungal biomasses exhibited the highest dye biosorption at pH 2.0. The Langmuir adsorption model appears to fit the dye biosorption better than the Freundlich model, with maximum dye uptake capacities ranging from 526 to 1111 mg/g, depending on the biomass used.     

Molecular Cloning,Characterization, and Dye-Decolorizing Ability of a Temperature- and pH-Stable Laccase from Bacillus subtilis X1

Laccases from fungal origin are typically unstable at high temperatures and alkaline conditions. This characteristic limits their practical applications. In this study, a new bacterial strain exhibiting laccase activity was isolated from raw fennel honey samples and identified as Bacillus subtilis X1. The CotA-laccase gene was cloned from strain X1 and efficiently expressed in Escherichia coli in a biologically active form. The purified recombinant laccase demonstrated an extensive pH range for catalyzing substrates and high stability toward alkaline pH and high temperatures. No loss of laccase activity was observed at pH 9.0 after 10 days of incubation, and approximately 21 % of the initial activity was detected after 10 h at 80 °C. Two anthraquinonic dyes (reactive blue 4 and reactive yellow brown) and two azo dyes (reactive red 11 and reactive brilliant orange) could be partially decolorized by purified laccase in the absence of a mediator. The decolorization process was efficiently promoted when methylsyringate was present, with more than 90 % of color removal occurring in 3 h at pH 7.0 or 9.0. These unusual properties indicated a high potential of the novel CotA-laccase for industrial applications.     

Decolorization of Synthetic Dyes by Crude and Purified Laccases from Coprinus comatus Grown Under Different Cultures: The Role of Major Isoenzyme in Dyes Decolorization

Coprinus comatus laccase isoenzyme induction and its effect on decolorization were investigated. The C/N ratio, together with aromatic compounds and copper, significantly influenced laccase isoenzyme profile and enzyme activity. This fungus produced six laccase isoenzymes in high-nitrogen low-carbon cultures but much less in low-nitrogen high-carbon (LNHC) cultures. The highest laccase level (3.25 IU/ml), equivalent to a 12.6-fold increase compared with unsupplemented controls (0.257 IU/ml), was recorded after 13 days in LNHC cultures supplemented with 2.0 mM 2-toluidine. Decolorization of twelve synthetic dyes belonging to anthraquinone, azo, and triphenylmethane dyes, by crude laccases with different proportion of isoenzymes produced under selected culture conditions, illustrated that the LacA is the key isoenzyme contributed to dyes decolorization especially in the presence of 1-hydroxybenzotriazol, which was further confirmed by dyes decolorization with purified LacA in the same condition. The crude laccase only was able to decolorize over 90 % of Reactive Brilliant Blue K-3R, Reactive Dark Blue KR, and Malachite Green, and higher decolorization for broader spectrum of synthetic dyes was obtained in presence of redox mediator, suggesting that C. comatus had high potential to decolorize various synthetic dyes as well as the recalcitrant azo dyes.     

Screening thermotolerant white-rot fungi for decolorization of wastewaters

To select a thermotolerant fungal strain for decolorization of wastewaters, ligninolytic enzyme production (lignin peroxidase, manganese peroxidase [MnP], and laccase), decolorization, and removal of total phenol and chemical oxygen demand (COD) were detected. Thirty-eight fungal strains were studied for enzyme production at 35 and 43°C on modified Kirk agar medium including 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and MnCl₂. Thirteen strains grew on manganese-containing agar and provided green color on ABTS-containing agar plates under culture at 43°C. Decolorization of wastewater from alcohol distillery (WAD) by these strains was compared under static culture at 43°C, and Pycnoporus coccineus FPF 97091303 showed the highest potential. Thereafter, immobilized mycelia were compared with free mycelia for WAD decolorization under culture conditions of 43°C and 100 rpm. The immobilized mycelia on polyurethane foam enhanced the ligninolytic enzyme production as well as total phenol and color removal. At about the same COD removal, MnP and laccase produced by immobilized mycelia were 2 and 19 times higher than by free mycelia; the simultaneous total phenol and color removal were 3.1 and 1.5 times higher than the latter. Moreover, decolorization of synthesis dye wastewater was carried out at 43°C and 100 rpm. More than 80% of 300 mg/L of reactive blue-5 was decolorized by the immobilized mycelia within 1 to 2 d for four cycles.     

Comparative study of amidase production by free and immobilized Escherichia coli cells

Escherichia coli NCIM 2569 was evaluated for its potential for amidase production under submerged fermentation. Among the various amide compounds screened, maximum substrate specificity and enzyme yield (8.1 U/mL) were obtained by using 1% acetamide. Fermentation was carried out at 30°C in shake-flask culture under optimized process conditions. A maximum of 0.52 U/mL of intracellular amidase activity was also obtained from cells incubated for 24 h. Studies were also performed to elucidate the optimal conditions (gel concentration, initial biomass, curing period of beads, and calcium ion concentration in the production medium) for immobilization of whole cells. By using E. coli cells entrapped in alginate, a maximum of 6.2 U/mL of enzyme activity was obtained after 12 h of incubation under optimized conditions. Using the immobilized cells, three repeated batches were carried out successfully, and 85% of the initial enzyme activity was retained in the second and third batches. The study indicated that the immobilized E. coli cells offered certain advantages such as less time for maximum enzyme production, more stability in the enzyme production rate, and repeated use of the biocatalyst.     

A Novel Kinetic Approach for Photocatalytic Degradation of Azo Dye with CdS and Ag/CdS Nanoparticles Fixed on a Cement Bed in a Continuous‐Flow Photoreactor

Azo dyes are one of the synthetic dyes that have been used in many textile industries. Azo dye and their intermediate products are toxic, carcinogenic, and mutagenic to aquatic life. Removal of azo dyes is one of the main challenges before releasing the wastes discharged by textile industries. Photocatalytic degradation of azo dyes by nanoparticles is one of the environment‐friendly methods used for the removal of dyes from textile effluents. Therefore, this study focused on degradation of azo dye, Direct Red 264. Photocatalytic degradation of DR 264 azo dye was investigated using CdS and Ag/CdS nanoparticles immobilized on a cement bed in a continuous‐flow photoreactor under UV‐C exposure. The effect of the parameters of type and mass of catalyst, temperature, flow rate, dye concentration, and light intensity were evaluated for azo dye removal. Under optimal conditions, photocatalytic degradation of DR 264 azo dye using Ag/CdS nanoparticles immobilized on a cement bed in a continuous‐flow photoreactor obtained an efficiency of 99.99%. A developed kinetic model was proposed based on the intrinsic elementary reactions. The proposed model is in a good agreement with the Langmuir–Hinshelwood (L–H) equation. The pseudo–steady‐state approximation has considered for the concentration of hydroxyl radicals associated with the L–H model under certain conditions and explains consistently the dependence of the apparent kinetic parameter, k_obs (the reaction rate constant), and K_R (the adsorption equilibrium constant) with the light intensity. Based on the model, k_obs for Ag/CdS was greater than the CdS nanoparticles.     

Aerobic Biodegradation Pathway for Remazol Orange by Pseudomonas aeruginosa

Removal of azo dyes from effluent generated by textile industries is rather difficult. Azo dyes represent a major class of synthetic colorants that are mutagenic and carcinogenic. Pseudomonas aeruginosa grew well in the presence of Remazol Orange (RO) and was able to decolorize and degrade it. In the present study, the decolorization and degradation efficiency using single culture P. aeruginosa with RO and textile wastewaters is studied. The elucidation of decolorization pathway for P. aeruginosa is of special interest. The degradation pathway and the metabolic products formed during the degradation were also predicted with the help of high performance liquid chromatography, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy analysis. The data show the cleavage of the azo dye RO to form both methyl metanilic acid and 4-aminobenzoic acid after decolorization and finally to oxidation forms benzoic acid, alkenes, aldehydes, and alkynes. The organism was able to decolorize the dye RO and wastewater effectively to the maximum of 82.4% and 62%, respectively.     

Efficiency of a fixed-bed and a gas-lift three-column reactor for continuous production of ethanol by pectate-and alginate-immobilized Saccharomyces cerevisiae cells

Ethanol-tolerant and thermo-tolerant yeast strain Saccharomyces cerevisiae C11-3 cells immobilized in calcium pectate and calcium alginate gels were used for ethanol fermentation in a three-reactor system with a gradient temperature control. The fermentation process has been tested in a fixed-bed and a gas-lift arrangement. The gas-lift system was more efficient due to a better mass transport between the phases. Abrasion was more evident in calcium alginate particles, while calcium pectate beads were not significantly damaged. Two different concentrations of alginate were tested and calcium pectate gel was demonstrated to be more suitable as an immobilization material in comparison with calcium alginate due to its mechanical resistance and favourable diffusion parameters, providing an ethanol production of more than 7.5 g dm⁻³ h⁻¹ over a period of 630 h.     

Polysaccharide/Fe(III)-porphyrin hybrid film as catalyst for oxidative decolorization of toxic azo dyes: An approach for wastewater treatment

A hybrid film of chitosan/poly(vinyl alcohol)/cationic Fe(III)-porphyrin (Cht/PVA-FeTMPyP) was synthesized to act as a Fenton-like catalyst to decolorize methyl orange and methyl red azo dyes. The Cht/PVA-FeTMPyP film was characterized by different analytical and microscopy techniques, which indicated that the metalloporphyrin affects different properties of the hybrid film. Batch experiments revealed that the hybrid film exhibits enhanced catalytic activity towards dyes decolorization in the presence of H₂O₂ as compared to the “free” FeTMPyP. Fast decolorization rates as high as 90 min were observed for both azo dyes under mild conditions (pH 7 and room temperature), even at low concentrations of the catalyst in H₂O₂. After the decolorization, FTIR analysis showed that simple molecules are released as by-products. Moreover, the hybrid film performed well in cyclic runs without leaching out iron ions or losing its catalytic activity. All these features associated with its ease handling ranks the Cht/PVA-FeTMPyP hybrid film as a promising heterogeneous Fenton-like catalyst for the decomposition of azo dyes in water.     

Thermokinetic Comparison of Trypan Blue Decolorization by Free Laccase and Fungal Biomass

Free laccase and fungal biomass from white-rot fungi were compared in the thermokinetics study of the laccase-catalyzed decolorization of an azo dye, i.e., Trypan Blue. The decolorization in both systems followed a first-order kinetics. The apparent first-order rate constant, k ₁′, value increases with temperature. Apparent activation energy of decolorization was similar for both systems at ～22 kJ mol^?1, while energy for laccase inactivation was 18 kJ mol^?1. Although both systems were endothermic, fungal biomass showed higher enthalpy, entropy, and Gibbs free energy changes for the decolorization compared to free laccase. On the other hand, free laccase showed reaction spontaneity over a wider range of temperature (ΔT?=?40 K) as opposed to fungal biomass (ΔT?=?15 K). Comparison of entropy change (ΔS) values indicated metabolism of the dye by the biomass.     

Black Liquor Decolorization by Selected White-Rot Fungi

Five different strains of white-rot fungi have been tested for their ability to decolorize black liquor on plates and on solid-state fermentation using vermiculite as the solid inert support. Since the high salt concentration inhibited the growth of all fungi, the black liquor was dialyzed against distilled water prior to use. A preliminary step on plates was carried out to qualitatively determine the capacity of the fungal strains for black liquor decolorization. Out of the five fungi studied, Phanerochaete sordida, Pycnoporus sanguineus, and Trametes elegans exhibited the more conspicuous decolorization halos in malt extract medium, while the decolorization by all the strains was not evident when a defined culture medium was used. Cultures on solid-state fermentation using vermiculite as solid support were also tested, the liquid phase was malt extract or glucose-based medium and supplemented with different black liquor concentrations. Decolorization of black liquor was largely affected by the fungal strain, the concentration of black liquor, and the carbon source. The percentage of color removal ranged from 6.14% to 91.86% depending on the fungal strain and culture conditions. Maximal decolorization was observed in malt extract cultures after 60 cultivation days. Interestingly, decolorization in malt extract medium increased with increasing black liquor concentration. The highest decolorization value was achieved by Steccherinum sp. which reduced up to 91.86% the color of the black liquor in malt extract medium; this percentage is equivalent to 5.2 g L⁻¹ of decolorized black liquor, the highest value reported to date. Traditional technologies used for the treatment of black liquor are not always effective and may not to be an environmentally friendly process. Vermiculite–white-rot fungi systems are presented in this work as a promising efficient alternative for the treatment of black liquor.     

Affinity Covalent Immobilization of Glucoamylase onto ρ-Benzoquinone-Activated Alginate Beads: II. Enzyme Immobilization and Characterization

A novel affinity covalent immobilization technique of glucoamylase enzyme onto ρ-benzoquinone-activated alginate beads was presented and compared with traditional entrapment one. Factors affecting the immobilization process such as enzyme concentration, alginate concentration, calcium chloride concentration, cross-linking time, and temperature were studied. No shift in the optimum temperature and pH of immobilized enzymes was observed. In addition, K _m values of free and entrapped glucoamylase were found to be almost identical, while the covalently immobilized enzyme shows the lowest affinity for substrate. In accordance, V _m value of covalently immobilized enzyme was found lowest among free and immobilized counter parts. On the other hand, the retained activity of covalently immobilized glucoamylase has been improved and was found higher than that of entrapped one. Finally, the industrial applicability of covalently immobilized glucoamylase has been investigated through monitoring both shelf and operational stability characters. The covalently immobilized enzyme kept its activity over 36 days of shelf storage and after 30 repeated use runs. Drying the catalytic beads greatly reduced its activity in the beginning but recovered its lost part during use. In general, the newly developed affinity covalent immobilization technique of glucoamylase onto ρ-benzoquinone-activated alginate carrier is simple yet effective and could be used for the immobilization of some other enzymes especially amylases.     

Strategies for Enhancing Laccase Yield from <Emphasis Type="Italic">Streptomyces psammoticus</Emphasis> and Its Role in Mediator-based Decolorization of Azo Dyes

Enhanced production of laccases from Streptomyces psammoticus in solid-state fermentation was carried out using two different strategies: laccase inducers and scale-up process. Laccase yield was enhanced by a wide range of aromatic inducers. The best inducer was pyrogallol, which yielded 116 U/g as compared to the control (55.4 U/g). Scale-up studies in packed bed bioreactor was performed at different aeration rates. Aeration at 1.5 vvm was identified as the optimum condition for laccase production (75.4 U/g) in the column bioreactor. The enzyme yield was enhanced further by combining the best conditions from the first two experiments. Fermentation was carried out in bioreactors in the presence of 1 mM pyrogallol, which resulted in 3.9-fold increase in laccase yield (215.6 U/g). The role of laccase in azo dye decolorization was evaluated in the presence of four different laccase mediators, at different concentrations. 1-Hydroxybenzotriazole (HOBT) proved to be the best mediator for S. psammoticus laccase and decolorized the azo dyes efficiently. Acid orange, Methyl orange, and Bismarck brown were decolorized at the rates of 86%, 71%, and 75% respectively, by HOBT.     

Screening and Immobilization <Emphasis Type="Italic">Burkholderia</Emphasis> sp. GXU56 Lipase for Enantioselective Resolution of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-Methyl Mandelate

Microorganisms producing lipase were isolated from soil and sewage samples and screened for enantioselective resolution of (R,S)-methyl mandelate to (R)-mandelic acid. A strain designated as GXU56 was obtained and identified as Burkholderia sp. Preparing immobilized GXU56 lipase by simple adsorption on octyl sepharose CL-4B, the optimum temperature was shifted from 40 °C (free lipase) to 50 °C (immobilized lipase), and the optimum pH was shifted from 8.0 (free lipase) to 7.2 (immobilized lipase). The immobilized enzyme displayed excellent stability in the pH range of 5.0–8.0, at the temperatures below 50 °C and in organic solvents compared with free enzyme. Enantioselectivity ratio for (R)-mandelic acid (E) was dramatically improved from 29.2 to more than 300 by applying immobilized lipase in the resolution of (R,S)-methyl mandelate. After five cycles of use of immobilized lipase, conversion and enantiomeric excess of (R)-mandelic acid were 34.5% and 98.5%, respectively, with enantioselectivity ratio for (R)-mandelic acid (E) of 230. Thus, octyl-sepharose-immobilized GXU56 lipase can be used as a bio-resolution reagent for producing (R)-mandelic acid.     

Decolorization and partial degradation of selected azo dyes by methanogenic sludge

The toxicity potential and decolorization of three acid azo dyes (Acid Orange 6, Acid Orange 7, and Acid Orange 52) by methanogenic granular sludge from an anaerobic expanded granular sludge bed reactor was assayed. Complete bioreduction was found for all three azo dyes. Sulfanilic acid and 4-aminoresorcinol were detected from the decolorization of Acid Orange 6, sulfanilic acid and 1-amino-2-naphtol were detected from the reduction of Acid Orange 7, and sulfanilic acid and N,N-dimethyl-1,4-phenylenediamine (DMP) were found to be intermediates of Acid Orange 52 degradation. Sulfanilic acid and 1-amino-2-naphtol were persistent in the anaerobic conditions, whereas 4-aminoresorcinol was completely mineralized by anaerobic sludge and DMP was transformed into 1,4-phenylenediamine. Enrichment cultures obtained via consecutive passages on basal medium with only azo dye as a carbon and an energy source seemed to be morphologically heterogeneous. Baculiform and coccus cells were found when viewed under a light microscope. Cocci were joined in chains. Because anaerobic sludge contains sulfate-reducing bacteria and therefore may generate sulfide, azo dyes were tested for chemical decolorization by sulfide to compare rates of chemical and biologic reduction.