Removal of phenols from wastewater by soluble and immobilized tyrosinase

An enzymatic method for removal of phenols from industrial wastewater was investigated. Phenols in an aqueous solution were removed after treatment with mushroom tyrosinase. The reduction order of substituted phenols is catechol > p-cresol > p-chlorophenol > phenol > p-methoxyphenol. In the treatment of tyrosinase alone, no precipitate was formed but a color change from colorless to dark-brown was observed. The colored products were removed by chitin and chitosan which are available abundantly as shellfish waste. In addition, the reduction rate of phenols was observed to be accelerated in the presence of chitosan. Tyrosinase, immobilized by using amino groups in the enzyme on cation exchange resins, can be used repeatedly. By treatment with immobilized tyrosinase, 100% of phenol was removed after 2 h, and the activity was reduced very little even after 10 repeat treatments. (c) 1993 John Wiley & Sons, Inc.     

Bioremoval of Cyanide and Phenol from Industrial Wastewater: An Update

In nature, phenols and cyanides are produced by certain microbes and plants. Phenols are antioxidants found in almost all plants, and cyanides are important components of lima beans, almonds, and cassava. Their presence in small amounts may not upset the environment, but their large-scale production, wide applicability, and unrestricted release by the industries makes them widespread and important pollutants. Phenols and cyanides can be recovered/removed from wastewater streams using various physicochemical techniques practiced commercially. Lack of complete mineralization, cost-effectiveness, and release of secondary by-products are amongst a few of the major considerations that limit the installation of such processes. Biological removal of such pollutants from industrial waste has gained momentum in recent years, as they promise to surpass the major drawbacks laid by the physicochemical methods and can be practically carried out in all conditions. Presence of either cyanide or phenol is highly dangerous, and in the presence of both, the effect is compounded. The present review illustrates the various industries involved in the release of phenols, cyanides, or both; it summarizes the available technologies for their treatment and emphasizes recent advances and advantages of biological abatement of these pollutants.     

Enhanced chemiluminescence in the peroxidase-luminol-H2O2 system: Anomalous reactivity of enhancer phenols with enzyme intermediates

Phenols which markedly enhance chemiluminescence in the horseradish peroxidase catalysed oxidation of luminol by hydrogen peroxide show anomalously high reactivity (by factors of ～10² compared with published Hammett correlations) in the reduction of the enzyme intermediates, Compound I and Compound II. The results support the hypothesis that efficient production of phenoxy radicals from phenols is a necessary criterion for chemiluminescence enhancer action.     

Water purification through bioconversion of phenol compounds by tyrosinase and chemical adsorption by chitosan beads

Enzymatic removal of various phenol compounds from artificial wastewater was undertaken by the combined use of mushroom tyrosinase (EC 1.14.18.1) and chitosan beads as function of pH value, temperature, tyrosinase dose, and hydrogen peroxide-to-substrate ratio. Chitosan film incubated in a p-crersol+tyrosinase mixture had the main peaks at 400-470 nm assigned to chemically adsorbed quinone derivatives, which increased over the immersion time. These results indicate that removal of phenol compounds is caused by their tyrosinase-catalyzed oxidation to the corresponding quinone derivatives and the subsequent chemical adsorption on the chitosan film. The optimum conditions for quinone adsorption were determined to be pH 7 and 45 degrees C for p-cresol. Some alkyl-substituted phenol compounds were removed by adsorption of quinone derivatives enzymatically generated on the chitosan beads, and the % removal for p-cresol, 4-ethylphenol, 4-n-propylphenol, 4-n-butylphenol, and p-chlorophenol went up to 93%. In addition, 4-tert-butylphenol underwent tyrosinase-catalyzed oxidation in the presence of hydrogen peroxide. This procedure was applicable to removal of chlorophenols and alkyl-substituted phenols.     

Dephenolization of industrial wastewaters catalyzed by polyphenol oxidase

A new enzymatic method for the removal of phenols from industrial aqueous effluents has been developed. The method uses the enzyme polyphenol oxidase which oxidizes phenols to the corresponding o-quinones; the latter then undergo a nonenzymatic polymerization to form water-insoluble aggregates. Therefore, the enzyme in effect precipitates phenols from water. Polyphenol oxidase has been found to nearly completely dephenolize solutions of phenol in the concentration range from 0.01 to 1.0 g/L. The enzymatic treatment is effective over a wide range of pH and temperature; a crude preparation of polyphenol oxidase (mushroom extract) is as effective as a purified, commercially obtained version. In addition to phenol itself, polyphenol oxidase is capable of precipitating from water a number of substituted phenols (cresols, chlorophenols, naphthol, etc.). Also, even pollutants which are unreactive towards polyphenol oxidase can be enzymatically coprecipitated with phenol. The polyphenol oxidase treatment has been successfully used to dephenolize two different real industrial waste-water samples, from a plant producing triarylphosphates and from a coke plant. The advantage of the polyphenol oxidase dephenolization over the peroxidase-catalyzed one previously elaborated by the authors is that the former enzyme uses molecular oxygen instead of costly hydrogen peroxide (used by peroxidase) as an oxidant.     

Polyethylene glycol improves phenol removal by immobilized turnip peroxidase

Purified peroxidase from turnip (Brassica napus L. var. esculenta D.C.) was immobilized by entrapment in spheres of calcium alginate and by covalent binding to Affi-Gel 10. Both immobilized Turnip peroxidase (TP) preparations were assayed for the detoxification of a synthetic phenolic solution and a real wastewater effluent from a local paints factory. The effectiveness of phenolic compounds (PC's) removal by oxidative polymerization was evaluated using batch and recycling processes, and in the presence and in the absence of polyethylene glycol (PEG). The presence of PEG enhances the operative TP stability. In addition, reaction times were reduced from 3h to 10min, and more effective phenol removals were achieved when PEG was added. TP was able to perform 15 reaction cycles with a real industrial effluent showing PC's removals >90% PC's during the first 10 reaction cycles. High PC's removal efficiencies (>95%) were obtained using both immobilized preparations at PC's concentrations <1.2mM. Higher PC's concentrations decreased the removal efficiency to 90% with both preparations after the first reaction cycle, probably due to substrate inhibition. On the other hand, immobilized TP showed increased thermal stability when compared with free TP. A large-scale enzymatic process for industrial effluent treatment is expected to be developed with immobilized TP that could be stable enough to make the process economically feasible.     

Effect of temperature and pH on phenol removal using purified Coprinus cinereus peroxidase

The removal of phenol by peroxidase-catalysed polymerization was examined using purified Coprinus cinereus peroxidase. The phenol removal efficiency increased with a decrease in the reaction temperature over the range of 0–70 °C, though only a trace of enzyme activity with 4-aminoantipyrine (4-AAP), phenol and hydrogen peroxide was found at 0 °C. The optimum pH value for phenol removal was 9.0, while the enzyme expressed maximum activity at pH 7.5 in the presence of 4-AAP, phenol and hydrogen peroxide. By measuring residual enzyme activity in the polymerizing reaction mixture, it was shown that enzyme inactivation by free radicals was more suppressed at 0 °C than at 40 °C and that the adsorption of the enzyme on the polymerized precipitate was more suppressed at pH 9.0 than that at pH 7.5.     

Phenol removal from aqueous solutions by peroxidase extracted from horseradish

Horseradish peroxidase (HRP) is one of the most recently used enzymes in the process of enzymatic phenol removal. It has a catalytic ability over a broad range of pH, temperature and contaminant concentrations. In this study we revealed the possibility of successful use the crude peroxidase obtained from horseradish roots for the phenol removal from aqueous solutions in the presence of the low molecular polyethylene glycol (PEG 300) at room temperature (20°C) and pH 7.2. Reaction was monitored by direct measuring of the absorbance changes in a samples taken at certain time intervals from the reaction mixture. At the first time PEG 300 was shown to be a more stabilizing effect on crude HRP and provided a higher phenol removal in comparison with PEG 3350. Crude HRP used in these study demonstrated a greater resistance on phenol and hydrogen peroxide inactivation that allowed a higher phenol removal. The highest phenol removal was achieved when the concentration of PEG 300, phenol and hydrogen peroxide were 300 mg/L, 2.0 and 2.5 mM, respectively.     

Effect of reaction conditions on phenol removal by polymerization and precipitation using Coprinus cinereus peroxidase

The quantitative relationships between removal efficiency of phenol and reaction conditions were investigated using Coprinus cinereus peroxidase. The most effective ratio of hydrogen peroxide to phenol was nearly 1/1 (mol/mol) at an adequate enzyme dose. 12.2 U of the enzyme was needed to remove 1 mg of phenol when our peroxidase preparation was used. At an insufficient peroxidase dose, the optimum pH value was 9.0, and lowering the reaction temperature led to the improvement of removal efficiency. At an excess peroxidase dose, almost 100% removal of phenol was obtained over a wide range of pH (5-9) and temperature (0-60 degrees C). Despite the presence of culture medium components, it was shown that Coprinus cinereus peroxidase had the same phenol polymerization performance as horseradish peroxidase or Arthromyces ramosus peroxidase.     

The Role of Micro-organisms in Waste Tip-lagoon Systems Purifying Coke-oven Effluents

S ummary . The population of aerobic bacteria present in the waters of a tip-lagoon system being used to purify a coke-oven effluent has been investigated. Though organisms capable of degrading phenol were detected, the total bacterial population was low, mainly due to a deficiency of orthophosphate and lack of aeration. Phenols can be removed from coke-oven effluents by allowing them to percolate through columns of material from colliery waste tips. Bacteria need not be present for this to occur though the presence of bacteria capable of degrading phenol were detected in the liquid coming from such columns. Only traces of thiocyanate are removed. If a biological filter can be developed, as on columns packed with gravel, better removal of phenols and thiocyanate occurs, but it is doubtful if bacteria play any significant role in the purification of coke-oven waste liquors percolating through large tips of colliery waste.     

A simple colorimetric method for determination of hydrogen peroxide in plant tissues

A simple colorimetric method for determination of hydrogen peroxide in plant materials is described. The method is based on hydrogen peroxide producing a stable red product in reaction with 4-aminoantipyrine and phenol in the presence of peroxidase. Plant tissues was ground with trichloroacetic acid (5% w/v) and extracts were adjusted to pH 8.4 with ammonia solution. Activated charcoal was added to the homogenate to remove pigments, antioxidants and other interfering substances. The colorimetric reagent (pH 5.6) consisted of 4-aminoantipyrine, phenol, and peroxidase. With this method, we have determined the hydrogen peroxide concentration in leaves of eight species which ranged from 0.2 to 0.8 μmol g⁻¹ FW. Changes in hydrogen peroxide concentration of Stylosanthes guianensis in response to heat stress are also analyzed using this method.     

Removal of α-naphthol and other phenolic compounds from polluted water by white radish (Raphanus sativus) peroxidase in the presence of an additive, polyethylene glycol

Role of white radish peroxidase has been investigated in the treatment of water contaminated with phenols, particularly α-naphthol. Water polluted with α-naphthol was treated with white radish peroxidase under various experimental conditions. The treatment of α-naphthol polluted water by this enzyme in presence of polyethylene glycol enhanced its removal. Studies carried out in absence of polyethylene glycol showed only 36% of α-naphthol removal however, 96% of it was removed in presence of 0.1 mg/mL of polyethylene glycol in 100 mM sodium phosphate buffer, pH 6.5, and 0.75 mM H₂O₂ at 40°C. The other phenols oxidized and removed from waste water under similar experimental conditions were 18%, m-cresol; 30%, p-chlorophenol; 62%, p-bromophenol; 20%, benzyl alcohol; 21%, quinol; 38%, 2,6-dichlorophenol; 13%, 2,4-dichlorophenol; and 2%, native phenol. Mixtures of different phenolic compounds removed under identical treatment conditions were 63%, A; 40%, B; 52%, C; 41%, D; 72%, E; 66%, F; and 72%, G. Thus, peroxidase in presence of an additive, polyethylene glycol could be a suitable tool for the removal of phenolic compounds from industrial effluents.     

Enzymatic removal of phenols from aqueous solutions by Coprinus cinereus peroxidase and hydrogen peroxide.

The fungal enzyme Coprinus cinereus peroxidase (CIP) can be used for the removal of toxic phenols from water. After treating aqueous solutions of phenols with CIP and H2O2 the phenols polymerized and precipitated. The decrease in phenol concentration was investigated for 10 different phenols. At neutral pH, the investigated phenols were in general removed with high efficiency.     

Biotechnological applications of peroxidases

Peroxidases are widely distributed in nature. Reduction of peroxides at the expense of electron donating substrates, make peroxidases useful in a number of biotechnological applications. Enzymes such as lignin peroxidase and manganese peroxidase, both associated with lignin degradation, may be successfully used for biopulping and biobleaching in the paper industry, and can produce oxidative breakdown of synthetic azo dyes. Oxidative polymerization of phenols and aromatic amines conducted by horseradish peroxidase (HRP) in water and water-miscible organic solvents, may lead to new types of aromatic polymers. Site directed mutagenesis of HRP has been used to improve the enantioselectivity of arylmethylsulfide oxidations. Peroxidase has a potential for soil detoxification, while HRP as well as soybean and turnip peroxidases have been applied for the bioremediation of wastewater contaminated with phenols, cresols, and chlorinated phenols. Peroxidase based biosensors have found use in analytical systems for determination of hydrogen peroxide and organic hydroperoxides, while co-immobilized with a hydrogen peroxide producing enzyme, they can be used for determination of glucose, alcohols, glutamate and choline. Peroxidase has also been used for practical analytical applications in diagnostic kits, such as quantitation of uric acid, glucose, cholesterol, lactose, and so on. Enzyme linked immunorbent assay (ELISA) tests on which peroxidase is probably the most common enzyme used for labeling an antibody, are a simple and reliable way of detecting toxins, pathogens, cancer risk in bladder and prostate, and many other analytes. Directed evolution methods, appear to be a valuable alternative to engineer new catalyst forms of plant peroxidases from different sources to overcome problems of stability and to increase thermal resistance.     

Application of immobilized horseradish peroxidase onto modified acrylonitrile copolymer membrane in removing of phenol from water

A non-modified and modified with NaOH and ethylenediamine ultrafiltration membranes prepared from AN copolymer have been used as carriers for the immobilization of horseradish peroxidase (HRP) enzyme. The amount of bound protein onto the membranes and the activity of the immobilized enzyme have been investigated as well as the pH and thermal optimum, and the thermal stability of the free and immobilized HRP. The experiments have proved that the modified membrane is a better support for the immobilization of HRP enzyme. The latter has shown a greater thermal stability than the free enzyme.A possible application has been studied for reducing phenol concentration in water solutions through oxidation of phenol by hydrogen peroxide, in the presence of free and immobilized HRP enzyme on modified AN copolymer membranes. A higher degree of the phenol oxidation has been observed in the presence of the immobilized enzyme. A total removal of phenol has been achieved in the presence of immobilized HRP at concentration of the hydrogen peroxide 0.5 mmol L^?1 and concentration of the phenol in the model solutions within the interval 5–40 mg L^?1. A high degree of phenol oxidation (95.4%) has been achieved in phenol solution with 100 mg L^?1 concentration in the presence of hydrogen peroxide and immobilized HRP, which demonstrates the promising opportunity of using the enzyme for bioremediation of waste waters, containing phenol.The immobilized HRP has shown good operational stability. Deactivation of the immobilized enzyme to 50% of the initial activity has been observed after the 20th day of the enzyme operation.     

Enzymatic coupling of phenol vapors onto chitosan.

Phenols are important industrial chemicals, and because they can be volatile, also appear as air pollutants. We examined the potential of tyrosinase to react with the volatile phenol p-cresol. Three lines of evidence support the conclusion that volatile phenols react with tyrosinase and are coupled (i.e., chemisorbed) onto chitosan films. First, phenol-trapping studies indicated that p-cresol can be removed from vapors if the vapors are contacted with tyrosinase-coated chitosan films. Second, the ultraviolet absorbance of tyrosinase-coated chitosan films changes dramatically when they are contacted with cresol-containing vapors, whereas control films are unaffected by contacting with cresol vapors. Third, pressure measurements indicate that tyrosinase-coated chitosan films only react with cresol vapors if the oxygen cosubstrate is present. Additional studies demonstrate the potential of tyrosinase-coated chitosan films/membranes for the detection and removal of phenol vapors.     

Enhanced removal of three phenols by laccase polymerization with MF/UF membranes

Guaiacol, catechol, m-cresol are common phenolic compounds presented in various industrial effluents but difficult to be removed by conventional wastewater treatment schemes. To elucidate mechanisms of enhanced membrane removal by laccase polymerization, different MF and UF membranes were employed in a cross-flow module for phenol concentration of 5mM. With 2.98 IU/l of laccase applied at room temperature, guaiacol, catechol and m-cresol were polymerized to products of averaged molecular weight of 9600, 8350 and 5400 Da (Dalton), respectively. Methoxy and hydroxyl-substituted phenols (guaiacol and catechol) were polymerized better than methyl-substituted phenol (m-cresol) due to more stable free-radical containing intermediate structure induced by oxygen-containing methoxy and hydroxyl functional groups. Removal efficiencies for the un-reacted phenols were dependent on the molecular sizes (length and width), but were dependent on the molecular weight for the polymerized phenolic compounds. Flux was declined initially but reached steady state after 180 min of filtration, indicating these MF/UF membranes can be used for removal of these polymerized phenols without significant fouling. In addition, pretreatments by the inactivated laccase only caused further flux reduction without additional removal of phenols.     

Peroxidase catalyzed oxidation of naturally-occurring phenols and hardwood lignins

Horseradish peroxidase and hydrogen peroxide form phenoxy radicals from 4-substituted-2,6-dimethoxyphenols, milled wood lignin and alkali lignins. A number of factors governing this reaction are examined. Side chain cleavage to quinones is the principal disproportionation reaction of these radicals. Catalysis by UV light and inhibition by quinones is observed. Aerobic oxidation of phenols is catalyzed by small amounts of hydrogen peroxide. Lignin substrates are degraded by the same oxidation mechanism as are the simple phenolic substrates.     

Role of oxygen during horseradish peroxidase turnover and inactivation

Horseradish peroxidase catalyzed oxidation of phenol has been reinvestigated to determine the requirements of facile enzyme autoinactivation. Turnover of this peroxidase was monitored spectrophotometrically at 400 nm and found dependent on the concentration of phenol and hydrogen peroxide. The inactivation of the peroxidase required both substrates, phenol and H2O2, but surprisingly was also potentiated by molecular oxygen. Exclusion of diffusible superoxide or hydroxyl radicals had slight effect on product formation or loss of catalytic activity. A mechanism is proposed to explain the unanticipated role of oxygen during enzyme inactivation.     

Increased thermostability and phenol removal efficiency by chemical modified horseradish peroxidase

Horseradish peroxidase was modified by phthalic anhydride and glucosamine hydrochloride. The thermostabilities and removal efficiencies of phenolics by native and modified HRP were assayed. The chemical modification of horseradish peroxidase increased their thermostability (about 10- and 9-fold, respectively) and in turn also increased the removal efficiency of phenolics. The quantitative relationships between removal efficiency of phenol and reaction conditions were also investigated using modified enzyme. The optimum pH for phenol removal is 9.0 for both native and modified forms of the enzyme. Both modified enzyme could suffer from higher temperature than native enzyme in phenol removal reaction. The optimum molar ratio of hydrogen peroxide to phenol was 2.0. The phthalic anhydride modified enzyme required lower dose of enzyme than native horseradish peroxidase to obtain the same removal efficiency. Both modified horseradish peroxidase show greater affinity and specificity of phenol.