In Silico and in Vitro Physicochemical Screening of Rigidoporus sp. Crude Laccase-assisted Decolorization of Synthetic Dyes—Approaches for a Cost-effective Enzyme-based Remediation Methodology

The objective of this paper is to compare in silico data with wet lab physicochemical properties of crude laccase enzyme isolated from Rigidoporus sp. using wheat bran as solid substrate support towards dye decolorization. Molecular docking analysis of selected nine textile and non-textile dyes were performed using laccase from Rigidoporus lignosus as reference protein. Enzyme-based remediation methodology using crude enzyme enriched from solid state fermentation was applied to screen the effect of four influencing variables such as pH, temperature, dye concentration, and incubation time toward dye decolorization. The extracellular crude enzyme decolorized 69.8 % Acid Blue 113, 45.07 % Reactive Blue 19, 36.61 % Reactive Orange 122, 30.55 % Acid Red 88, 24.59 % Direct Blue 14, 18.48 % Reactive Black B, 16.49 % Reactive Blue RGB, and 11.66 % Acid Blue 9 at 100 mg/l dye concentration at their optimal pH at room temperature under static and dark conditions after 1 h of incubation without addition of any externally added mediators. Our wet lab studies approach, barring other factors, validate in silico for screening and ranking textile dyes based on their proximity to the T1 site. We are reporting for the first time a combinatorial approach involving in silico methods and wet lab-based crude laccase-mediated dye decolorization without any external mediators.     

Oxidation of azo dye Orange II with hydrogen peroxide catalyzed by 5,10,15,20-tetrakis[4-(diethylmethylammonio)phenyl]porphyrinato-cobalt(II)tetraiodide in aqueous solution

The catalytic oxidation of the azo dye Orange II by hydrogen peroxide in aqueous solution has been investigated using 5,10,15,20-tetrakis-[4-(diethylmethylammonio)phenyl]porphyrinato-cobalt(II) tetra iodide 1as catalyst. The oxidation reaction was followed by recording the UV–vis spectra of the reaction mixture with time at λ_max = 485 nm. The factors that may influence the oxidation of Orange II, such as the effect of reaction temperature, concentration of catalyst, hydrogen peroxide and orange II have been studied. The results of total organic carbon analysis showed 52% of dye mineralization under mild reaction conditions. Residual organic compounds in the reaction mixture were identified by using Gas chromatography-mass spectrometry. The decolorization rate and mineralization of the dye has been found to increase with increase of catalyst concentration and reaction temperature. The rate of dye oxidation decreased with increasing the concentration of dye, H₂O₂ and at higher pH than 9. Radical scavenging measurement indicated that decolorization of Orange II by H₂O₂/cobalt (II) porphyrin complex 1 involved the formation of hydroxyl radicals as the active species.     

Decolorization of Remazol Brilliant Blue R by a Purified Laccase of <Emphasis Type="Italic">Polyporus brumalis</Emphasis>

A white rot basidiomycete Polyporus brumalis has been reported to induce two laccase genes under degradation conditions of dibutylphthalate. When this fungus was grown in a minimal medium, one laccase enzyme was detected by the native polyacrylamide gel electrophoresis. A laccase was purified through ammonium sulfate precipitation and ion exchange chromatography, and the estimated molecular weight was 70 kDa. The optimum pH and temperature of the purified laccase was pH 4.0 and 20 °C, respectively. The K _m value of the enzyme was 685.0 μM, and the V _max was 0.147 ODmin⁻¹ unit⁻¹ for o-tolidine. Purified laccase showed effective decolorization of a dye, Remazol Brilliant Blue R (RBBR), without any laccase mediator. However, this effect was reduced by a laccase inhibitor, kojic acid, which confirmed that the laccase was directly involved in the decolorization of RBBR.     

Key Factors Regarding Decolorization of Synthetic Anthraquinone and Azo Dyes

The factors affecting decolorization of anthraquinone dye represented by Reactive Blue 4 (RB4) and azo dye represented by Methyl Orange (MO) were studied in batch experiments under mesophilic (35 °C) and thermophilic (55 °C) anaerobic conditions. The results indicated differences in decolorization properties of the dyes with different chromophore structures. In abiotic conditions, MO could be decolorized by a physicochemical reaction when it was sterilized at 121 °C together with sludge cells or glucose. RB4 only showed absorption onto the cell mass. The presence of a redox mediator accelerated the decolorizing reaction when supplied together with glucose in the presence of sterilized sludge cells. In biotic conditions, the results indicated that the biological activity of microorganisms was an important factor in decolorization. The main factor involved in decolorization was the conversion of cosubstrate as electron donor, which reacted with dye as an electron acceptor in electron transfer. Redox mediators, anthraquinone-2-sulfonic acid, and anthraquinone could accelerate decolorization even if a small amount (0.2 mM) was applied. On the other hand, a high concentration of redox mediator (1.0 mM) had an inhibitory effect on decolorization especially under thermophilic conditions. In addition, the decolorization of dye was accelerated by increasing treatment temperature, as shown in biotic treatments. Based on these results, increasing the treatment temperature could be used to improve the decolorizing process of textile dye wastewater treatment, especially for recalcitrant dyes such as anthraquinone.     

An effective nanocomposite of polyaniline and ZnO: preparation,characterizations, and its photocatalytic activity

The polyaniline/zinc oxide (PANI/ZnO) nanocomposites were prepared by in situ polymerization of aniline monomer with ZnO nanomaterials and applied as a photocatalyst for the degradation of methylene blue (MB) dye. The morphological observations elicited the agglomerations of PANI sheets which occurred due to the interaction between PANI and ZnO nanomaterials in PANI/ZnO nanocomposites. As compared to pristine PANI, the UV–vis spectra exhibited that the absorption peak of π–π* transitions considerably shifted to higher wavelength at 360 nm from 325 nm in the nanocomposites. The photocatalytic activity results indicated the substantial degradation of MB dye by ~76% over the surface of PANI/ZnO nanocomposite catalyst under light illumination. The PANI/ZnO nanocomposites showed three times higher photocatalytic activity to MB dye degradation compared to pristine PANI might due to high photogenerated electron (ē)–hole (h⁺) pairs charge separation.     

Production and Characterization of a Novel Laccase with Cold Adaptation and High Thermal Stability from an Isolated Fungus

A new white-rot fungus SYBC-L1, which could produce an extracellular laccase, was isolated from a decayed Elaeocarpus sylvestris. The strain was identified as Pycnoporus sp. SYBC-L1 according to the morphological characteristics and ribosomal ITS1-5.8S-ITS2 RNA genomic sequence analysis. The highest laccase activity of 24.1 U ml⁻¹, which was approximately 40-fold than that in basal medium, was achieved in optimal culture medium in submerged fermentation. The laccase produced by Pycnoporus sp. SYBC-L1 was not only a cold adaptation enzyme with a relative catalytic activity of 30.2% at 0°C but also a high thermostable enzyme. The half-lives at 60, 70 and 80°C were 85.5, 37.2, and 2.6 h, respectively. The laccase could effectively decolorize weak acid blue AS and diamond black PV up to 88% and 74.7%, respectively, within 2 h in the absence of any redox mediators. The results suggested Pycnoporus sp. SYBC-L1 was a potential candidate for laccase production and industrial application.     

Characterization of a Recombinant Laccase B from Trametes hirsuta MX2 and Its Application for Decolorization of Dyes

Trametes hirsuta is able to secrete laccase isoenzymes including constitutive and inducible forms, and has potential application for bioremediation of environmental pollutants. Here, an inducible group B laccase from T. hirsuta MX2 was heterologously expressed in Pichia pastoris, and its yield reached 2.59 U/mL after 5 days of methanol inducing culture. The optimal pH and temperature of recombinant laccase (rLac1) to 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were 2.5 and 60 °C, respectively. Metal ions showed different effect on rLac1 which Mg²⁺, Cu²⁺, and K⁺ increased enzyme activity as their concentration increased, whereas Zn²⁺, Na⁺, and Fe²⁺ inhibited enzyme activity as their concentration increased. rLac1 showed good tolerance to organic solvents, and more than 42% of its initial activity remained in 10% organic solvents. Additionally, rLac1 exhibited a more efficient decolorization ability for remazol brilliant blue R (RBBR) than for acid red 1 (AR1), crystal violet (CV), and neutral red (NR). Molecular docking results showed RBBR has a stronger binding affinity with laccase than other dyes by interacting with substrate binding cavity of enzyme. The results indicated rLac1 may be a potential candidate for dye removal from textile wastewater.     

3,4-Ethylenedioxythiophene (EDOT)-based π-conjugated oligomers: Facile synthesis and excited-state properties

A facile synthetic method to produce soluble 3,4-ethylenedioxythiophene (EDOT) oligomers was developed by using iron (III) nitrate nonahydrate as the oxidizing reagent. Two EDOT oligomers, i.e. octomers and octodecamers were obtained as the major products. Their UV–vis absorption, fluorescence at room temperature and 77 K, and triplet transient difference absorption have been measured in CH₂Cl₂ solutions or glassy solutions. With increased conjugation length from EDOT octomers (A) to EDOT octodecamers (B), the UV–vis absorption, fluorescence and the triplet transient difference absorption band shift to longer wavelength. Both the oligomers also exhibit reasonably high efficiency to generate singlet oxygen.     

Synthesis and characterization of ZnO-doped cupric oxides and evaluation of their photocatalytic performance under visible light

ZnO–CuO binary oxide photocatalysts were synthesized by the liquid phase coprecipitation method. The catalysts were characterized by X-ray diffraction, transmission electron microscopy and UV–vis spectroscopy. The photocatalytic activity of the ZnO–CuO nanocomposites was estimated on the basis of decoloration of methyl orange dye under visible light. The effects of parameters such as calcining temperature, amount of catalyst and pH on the photocatalytic degradation efficiency of methyl orange solutions were investigated in detail. The maximum photocatalytic activity was obtained on ZnO–CuO nanocomposites with a calcining temperature of 350 °C, using a catalyst amount of 0.056 g/L and a pH of 7.5. The visible light-driven capability of ZnO–CuO nanocomposites is much better than that of commercially available TiO₂ photocatalysts under comparable conditions.     

Reactive Blue 4 Decolorization under Mesophilic and Thermophilic Anaerobic Treatments

Anaerobic decolorization of anthraquinone dye represented by Reactive Blue 4 (RB4) was studied to evaluate the factors involved in dye-reducing behaviors such as dye concentration, co-substrate, treatment temperature, salt content, and dye-reducing microbial consortia. The experiment was conducted using digested sludge treated under mesophilic (35 °C) and thermophilic (55 °C) conditions. The results indicated that the thermophilic treatment gave higher potential for this dye decolorization compared with the mesophilic one. A reduced form of RB4 did not show an auto-oxidizing reaction but treated RB4 dye was shown in light yellow color, the intensity of which was related to the initial concentration of the dye used in the treatments. Starch, which showed similar decolorizing efficiency under thermophilic conditions, could be used as a co-substrate instead of glucose for the purpose of operating cost reduction. Due to the high content of salt contained in dye wastewater, the effect of salt (NaCl) was investigated. Results showed that decolorization could be accelerated with a concentration of NaCl lower than 200 mM, but the decolorization was inhibited by high concentrations of salt. The presence of RB4 inhibited methane productivity, while total organic carbon (TOC) reduction was similar to control, without dye addition. Increasing the temperature accelerated the decolorizing potential and TOC reduction. The evaluation of dye-reducing microbial consortia was done with acidogen and methanogen inhibitors which acidogenesis microorganism was dominant in RB4 decolorization.     

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.     

Decolorization and mineralization of textile dyes at solution bulk by heterogeneous nanophotocatalysis using immobilized nanoparticles of titanium dioxide

Nanophotocatalysis using nanostructured semiconductors constitute one of the emerging technologies for destructive oxidation of organics such as dyes. This paper deals with the decolorization and mineralization of reactive dyes by heterogeneous nanophotocatalysis using an immobilized TiO₂ nanoparticle photocatalytic reactor. A simple and effective method was used to immobilization of titanium dioxide nanoparticles. Reactive Orange 107 (RO 107, sulphatoethylsulphonyl reactive dye) and Reactive Red 152 (RR 152, monochlorotriazine reactive dye) were used as model compounds. UV–vis and ion chromatography (IC) analyses were employed to obtain the details of the photocatalytic degradation of the selected dyes. The effects of operational parameters such as H₂O₂, dye concentration, anions (NO₃⁻, Cl⁻, SO₄²⁻, HCO₃⁻ and CO₃²⁻) and pH were investigated. Formate, acetate and oxalate anions were detected as dominant aliphatic intermediates where, they were further oxidized slowly to CO₂. Nitrate, sulfate and chloride anions were detected as the photocatalytic mineralization of RO 107 and RR 152. Kinetics analysis indicates that the photocatalytic decolorization rates can usually be approximated zero-order model for RO 107 and first-order model for RR 152 dyes. Results show that the photocatalytic process occurred at solution bulk and the employment of optimal operational parameters may lead to complete decolorization and mineralization of dye solutions.     

Ion-beam irradiation effects on polyimide-UV–vis and infrared spectroscopic study

Ion-beam irradiation effects on polyimide, Kapton™, were studied with respect to optical and electronic properties. Stack films of Kapton™ (12.5 μm thick) were irradiated to various ion beams in air or vacuo at room temperature and subjected to ultraviolet–visible (UV–vis) spectroscopy, and change in absorbance and energy gap is discussed. The UV–vis absorption spectrum, which is assigned to the transition of electrons in benzene rings from π to π* orbital, upon He²⁺ (6 MeV/u) irradiation in air, shifted towards longer wavelength direction for all cases, and the shift was more obvious for higher linear energy transfer (LET) ion beams. The energy gap of the transition was estimated, and the H⁺ and He²⁺ ion beams caused little change in the transition energy gap Eg, while the heavier ions such as C⁶⁺ and Si¹⁴⁺ caused more significant decrease. This decrease is assumed to the structural changes around benzene rings, and the infrared spectroscopy revealed breakage in imide groups next to benzene ring in the repeating unit of polyimide.     

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.     

Effective removal of anionic dyes from aqueous solutions by novel polyethylenimine-ozone oxidized hydrochar (PEI-OzHC) adsorbent

The adsorption behavior of the anionic dyes Remazol Brilliant Blue R (RBBR) and Reactive Black 5 (RB5) from aqueous solutions by polyethylenimine ozone oxidized hydrochar (PEI-OzHC) was investigated. The adsorption capacities of both dyes increased with functionalization of PEI in the hydrochar adsorbent. The results of surface characterization (FTIR, BET, TGA, elemental analysis, and SEM) showed that PEI modification greatly enhanced the adsorbent surface chemistry with a slight improvement of adsorbent textural properties. In addition, the adsorption kinetics data showed an excellent adsorption efficiency as reflected in the high removal percentages of the anionic dyes. The Isotherm results indicated that RBBR and RB5 dye adsorption occurred via monolayer adsorption, and chemisorption was the rate-controlling step. The PEI-OzHC adsorbent possesses higher maximum Langmuir adsorption capacity towards RBBR (218.3 mg/g) than RB5 (182.7 mg/g). This increase in adsorption capacity is attributed to the higher number of functional groups in RBBR that interact with the adsorbent. This study reveals the potential use of adsorbents derived from pine wood hydrochar in municipal as well as industrial wastewater treatment. Furthermore, surface chemistry modification is proven as an effective strategy to enhance the performance of biomass-derived adsorbents.     

Fabrication of metal-organic framework Universitetet i Oslo-66 (UiO-66) and brown-rot fungus Gloeophyllum trabeum biocomposite (UiO-66@GT) and its application for reactive black 5 decolorization

Many various industries use synthetic dyes as their raw materials. These dyes have triggered environmental problems because of the occurring effluents, and one of the environmentally safe solutions for this problem is biodegradation through microorganisms. Reactive Black 5 (RB5) dye degradation was performed by utilizing a metal-organic framework Universitetet i Oslo-66 (UiO-66) and Gloeophyllum trabeum (GT) fungus biocomposite. The UiO-66@GT composite was fabricated by inoculating the fungal culture in flasks with the PDB medium that contained UiO-66. This biocomposite was applied to decolorize and degrade RB5 dye, while pure GT culture can decolorize about 36.47% in five days. The percentage of RB5 decolorization was shown to be increased with the addition of UiO-66; the composite could decolorize RB5 up to 72.55% after five days incubation period. Moreover, the optimum conditions for the 100% targeted rate of RB5 decolorization found by the Response Surface Methodology (RSM) are: initial RB5 concentration (72.54 mg L^-1), pH (6.53), and temperature (38.06 °C). Two novel metabolites from RB5 decolorization by the composite were detected based on LCMS-QTOF analysis and were used to propose a degradation pathway: 6-((1-amino-7,8-dihydroxy-6-sulfonaphthalen-2-yl) diazinyl) cyclohexa-2,4-dien-1-ide (m/z = 360) and 3,4-diamino-5,6-dihydroxy-1,2,7,8-tetrahydronaphthalene-2,7-disulfonic acid (m/z = 354).     

Decolorization of Reactive Black 5 and Reactive Blue 4 Dyes in Microbial Fuel Cells

Microbial fuel cells (MFCs) have potential to treat industrial wastewater containing organic compounds and simultaneously generate power. Organic compounds include textile dyes with various chromophore groups, which can be decolorized reductively by microorganisms under anaerobic conditions. In the present study, we examined the decolorization of Reactive Black 5 (RB5) azo dye and Reactive Blue 4 (RBL4) anthraquinone dye under open circuit potential in MFCs with graphite plate and graphite felt electrodes and a microbial consortium originally derived from bovine rumen fluid. RB5 dye was more than 90% decolorized in 120, 165, and 225 min at 50, 100, and 200 mg L^?1 concentrations, respectively. RBL4 dye at 50 and 100 mg L^?1 took 225 and 300 min to decolorize, while 200 mg L^?1 RBL4 dye was not decolorized at all. Under closed circuit conditions, decolorization increased with decrease in external load, whereas current generation increased with external resistance. The results demonstrate that the reductive cleavage of the chromophore was more rapid with RB5 than with RBL4.     

Photochemical and photophysical properties of lumazine in aqueous solutions

Lumazine (pteridine-2,4(1H,3H)-dione, LU) was investigated for its efficiency of singlet oxygen (¹O₂) production and quenching in aqueous solution. The quantum yield of ¹O₂ production (Φ_Δ) was determined by measurements of the ¹O₂ luminescence in the near-infrared upon continuous excitation of the sensitizer. Values of Φ_Δ are sensitive to the pH and were found to be 0.44 ± 0.01 and 0.080 ± 0.004 in acidic and alkaline media, respectively. The photochemical stability of LU was investigated under different pH conditions, in the presence and in the absence of O₂. The photochemical consumption of LU in aqueous solution at room temperature under irradiation at 350 nm was followed by UV–vis spectrophotometry and HPLC. Values of the quantum yields of LU disappearance are low, indicating that LU is rather photostable under physiological conditions.     

Studies of (3-mercaptopropyl)trimethoxylsilane and bis(trimethoxysilyl)ethane sol–gel coating on copper and aluminum

Berbamine, a naturally occurring isoquinoline alkaloid extracted from Berberis sp., is the active constituent of some Chinese herbal medicines and exhibits a variety of pharmacological activities. The effects of berbamine on the structure of bovine serum albumin (BSA) were investigated by circular dichroism, fluorescence and absorption spectroscopy under physiological conditions. Berbamine caused a static quenching of the intrinsic fluorescence of BSA, and the quenching data were analyzed by application of the Stern–Volmer equation. There was a single primary berbamine-binding site on BSA with a binding constant of 2.577 × 10⁴ L mol⁻¹ at 298 K. The thermodynamic parameters, enthalpy change (ΔH⁰) and entropy change (ΔS⁰) for the reaction were −76.5 kJ mol⁻¹ and −173.4 J mol⁻¹ K⁻¹ according to the van’t Hoff equation. The results showed that the hydrogen bond and van der Waals interaction were the predominant forces in the binding process. Competitive experiments revealed a displacement of warfarin by berbamine, indicating that the binding site was located at Drug sites I. The distance r between the donor (BSA) and the acceptor (berbamine) was obtained according to the Förster non-radiation energy transfer theory. The results of three-dimensional fluorescence spectra, UV–vis absorption difference spectra and circular dichroism of BSA in the presence of berbamine showed that the conformation of BSA was changed. The results provide a quantitative understanding of the effect of berbamine on the structure of bovine serum albumin, providing a useful guideline for further drug design.     

Label-free detection of specific DNA sequence-telomere using unmodified gold nanoparticles as colorimetric probes

A simple and sensitive label-free colorimetric detection of telomere DNA has been developed. It was based on the color change of gold nanoparticles (AuNPs) due to DNA hybridization. UV–vis spectra and transmission electron microscopy (TEM) were used to investigate the change of AuNPs. Under the optimized conditions, the linear range for determination of telomere DNA was 5.7 × 10⁻¹³ to 4.5 × 10⁻⁶ mol/L. The detection limit (3σ) of this method has decreased to pico-molar level.