Biodegradation of 2,4-dinitrophenol with laccase immobilized on nano-porous silica beads
© Dehghanifard et al.; licensee BioMed Central Ltd. 2013
Received: 1 December 2012
Accepted: 12 March 2013
Published: 1 April 2013
Many organic hazardous pollutants, including 2,4-dinitrophenol (2,4-DNP), which are water soluble, toxic, and not easily biodegradable make concerns for environmental pollution worldwide. In the present study, degradation of nitrophenols-contained effluents by using laccase immobilized on the nano-porous silica beads was evaluated. 2,4-DNP was selected as the main constituent of industrial effluents containing nitrophenols. The performance of the system was characterized as a function of pH, contact time, temperature, pollutant, and mediator concentrations. The laccase-silica beads were employed in a mixed-batch reactor to determine the degradation efficiency after 12 h of enzyme treatment. The obtained data showed that the immobilized laccase degraded more than 90% of 2,4-DNP within 12 h treatment. The immobilization process improved the activity and sustainability of laccase for degradation of the pollutant. Temperatures more than 50°C reduced the enzyme activity to about 60%. However, pH and the mediator concentration could not affect the enzyme activity. The degradation kinetic was in accordance with a Michaelis–Menten equation with Vmax and Km obtained as 0.25–0.38 μmoles/min and 0.13–0.017 mM, respectively. The stability of the immobilized enzyme was maintained for more than 85% of its initial activity after 30 days. Based on the results, it can be concluded that high resistibility and reusability of immobilized laccase on CPC-silica beads make it considerable choice for wastewater treatment.
Nitrophenols, categorized as priority pollutants, are one of the main common components which release from industrial effluents and deteriorate the quality of water resources. There are six possible dinitrophenol (DNP) forms and 2,4-Dinitrophenol (2,4-DNP) is the most important toxic and refractory pollutant . 2,4-DNP, a yellowish crystalline solid, has been used in manufacturing of pesticides, pharmaceuticals, production of dyes, explosive materials, and as an indicator for the detection of potassium and ammonium ions. Its entrance into the environment may occur from industrial wastewaters, accidental spills, or as an intermediate metabolite due to degradation of pesticides containing 2,4-DNP . For instance, wastewater from a dye manufacturing plant contained 3.2 mg/L DNP. Groundwater from a waste site that was once occupied by a factory that used DNP contained 30.6 mg DNP/L of water .
Several physical and chemical methods which have been used for the treatment of the nitrophenols pollutants are adsorption processes, chemical oxidation, precipitation, evaporation, and incineration. However, due to their problems in the application and economic issues, other relevant treatment methods have been studied. The ability of biological processes on the degradation of organic pollutants, due to their effective and safe performance in compare with chemical and physical treatment techniques, has been considered [4, 5]. Among them, the performance of white rots fungi for degradation a wide range of refractory organic pollutants, specially the phenolic compounds, via lignin-modifying enzymes, such as manganese (II)-dependent peroxidase, lignin peroxidase, and laccase (phenol oxidase) have been studied [6–8]. Although biological processes are efficient at low pollutant concentrations, their sensitivity to shock loads, require long hydraulic retention times and forming large amounts of solid residues make their feasible application with challenges . In an enzyme based process, however, some of these disadvantages can be omitted since enzymes can be applied to persistent materials, high and low contaminant concentration over a wide pH, temperature, and salinity range. The most recent research in this area has focused on the enzymatic process for the treatment of wastewater [10, 11].
Laccase (EC 126.96.36.199), a multi-copper oxidase enzyme, catalyzes the oxidation of variety of aromatic and inorganic substrates, mostly phenols, with simultaneous reduction of oxygen to water [12–14]. Among advantages of laccase application such as high efficiency, the main disadvantage is that it is often easily inactivated in oxidation process due to the wide range of process conditions (temperature, pH, etc.) and also its separation procedure, for reuse proposes, from the reaction system is difficult which limits the further industrial applications of laccase. An effective method for reuse and improving its stability is using enzyme immobilization technology . Studies showed that several types of supporters could be used in enzyme immobilization which included activated carbon, chitosan microspheres , polymeric carrier , polyacrylonitrile beads  and magnetic chitosan nanoparticles .
Although many previous studies used immobilized laccase to degrade organic pollutants such as chlorophenols , dyes , Poly Aromatic Hydrocarbons (PAHs) , however there has not been a reliable research on oxidation of nitrophenol compounds by immobilized laccase. The aim of this study is to investigate the feasibility of 2,4-DNP degradation in effluents by Trametes versicolor laccase immobilized on the controlled porosity carrier (CPC) silica beads.
Materials and methods
T. versicolor laccase, pre-silanized [with 3-aminopropyltriethoxysilane (APTES)] CPC silica beads, Glutaraldehyde solution (25%), and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and 2,4-DNP, acetonitrile and methanol (HPLC grade) were from Merck (Darmstadt, Germany).
Laccase immobilization on CPC-silica beads
Laccase was immobilized on pre-silanized silica beads according to the study of Champagne and Ramsay . An amount of 4 g of pre-silanized CPC-silica beads (355–600 mm in diameter, an average surface area of 42.1 m2/g, and a pore size of 37.5 nm) were immersed in degassed 2.5% glutaraldehyde (2.0 bar vacuum pressure for 2 h) in 0.1 M KH2PO4, pH 5.0, then placed in an enzyme solution (≈ 2.0 U/mL in 0.1 M KH2PO4 at pH 5.0) for more than 36 h at 4°C. Thereafter, beads were purred on a paper filter and washed three times with distilled water and twice with phosphate buffer (0.1 M KH2PO4, pH 5.0).
Laccase activity was measured at 420 nm by generation of ABTS˚ˉ radicals from the enzymatic oxidation of ABTS at 25°C using CECIL 8600 spectrophotometer. The assay mixture contained 0.2 mM ABTS, 100 mM sodium acetate buffer (pH 5.0), and the enzyme-containing sample . One unit of laccase activity (U) was defined as the amount of enzyme that formed 1 μmol ABTS per min. Protein concentration was measured as the absorbance at 280 nm and corrected for scattering effects with absorbance readings at 320 nm .
Degradation of 2,4-DNP by immobilized laccase
For determining the performance of immobilized enzyme on 2,4-DNP degradation, 0.5 g of CPC-silica beads with 50 ± 3.8 U of laccase/g (i.e. 1.18 ± 0.09 U/m2) were used in a batch reactor (Erlenmeyer 50 mL). Synthetic effluent consisted of 2,4-DNP with concentrations of 0.05, 0.1, and 0.15 M (in 0.1 M phosphate buffer, pH 4–6) and ABTS (as a mediator) of 1, 2, and 3 mM was added to each reactor. Temperature had been adjusted in range of 40–60°C in the shaker incubator (150 rpm). The sampling procedure (1 mL for every 2 h up to 12 h retention time) was done, and then equal volume of methanol (HPLC grade) was added in order to end the reaction process between enzyme and the pollutant. Samples were then filtered by 0.2 μm PTFE (Polytetrafluoroethylene) filters and filled in 5 mL dark vessels (sealed by parafilm tape) in 4°C before analysis.
Analysis and measurements
The concentration of 2,4-DNP in the reaction mixture was measured by high performance liquid chromatography (HPLC, CECIL 4100, USA) equipped with an UV–Visible Diode Array detector. Separation of compounds was obtained with a Nucleodur Sphinx RP (25.0 cm × 4.6 mm) column (MZ-1 PerfectSil, Germany) at a flow-rate of 1 ml/min. The chromatographic determination was performed by using a gradient in 10 min acetonitrile/ 0.5% acetic acid (50:50, v/v). All assays were carried out in triplicate and gave standard deviations lower than 5%.
Results and discussion
Characterization of CPC-silica beads
As shown in Figure 1C, it could be observed that the laccase enzyme was effectively immobilized on the surface of the beads due to high surface area which the beads provided. In particular the nano-scale pores and the surface properties provided the suitable prerequisites for enzyme immobilization . By comparison of Figure 1B and C, the immobilized laccase was obviously detectable.
Characteristics of free and immobilized laccase
Enzymatic degradation of 2,4-DNP
Temperature is an important variable to be considered in laccase-catalyzed reactions and has a double effect on enzymatic systems which are a change in the rate of reaction over time caused by thermal inactivation of the enzyme, and a change in the reaction rate due to Arrhenius effects . Results revealed that by increasing temperature from 40 to 60°C, the pollutant degradation significantly decreased from 91% to only 33%, while other parameters kept constant (optimum condition). In compared with the resistance of free enzyme on temperature, as previously noted, its remained activity could be explained with the immobilization process and improving laccase characteristics . Tavares et al. showed that the activity immobilized laccase on modified silica beads was remained for more than 6 h in 55°C and by increasing the temperature up to 70°C, the enzyme was almost deactivated .
Kinetics of 2,4-DNP degradation
Kinetic parameters for the immobilized laccase using 2,4-DNP as substrates
Substrate concentration (mM)
Stability of immobilized enzyme
In this study, the performance of the immobilized laccase for degradation of 2,4-DNP in aqueous solutions has been investigated. Results showed that the immobilized enzyme was able to effectively degrade 2,4-DNP as a pollutant in water resources. The optimum condition for the maximum degradation of 2,4-DNP (91%) was achieved on 12 h contact time and pH 5. The immobilized laccase had more resistibility to the pH and temperature changes, in compare with the free form. The improved characteristics of immobilized laccase on CPC-silica beads for stability and reusability could be considered as an advantage in wastewater treatment. Investigation of the enzymatic degradation of other phenolic compounds with several mediators in different pH and Temperature condition could be recommended.
Authors highly appreciated of Tehran University of Medical Sciences for their financially supports of the study.
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