Removal of benzene from wastewater via Fenton pre-treatment followed by enzyme catalyzed polymerization.

This study investigated the feasibility of a two-step process for the removal of benzene from buffered synthetic wastewater. Benzene is outside the scope of enzymatic removal. In order to remove it from wastewater using enzyme, its pretreatment by modified Fenton reaction was employed to generate the corresponding phenolic compounds. In the first phase, the optimum pH, H2O2 and Fe2+ concentrations and reaction time for the Fenton reaction were determined to maximize the conversion of benzene to phenolic compounds without causing significant mineralization. The pretreatment process was followed by oxidative polymerization of the phenolic compounds catalyzed by a laccase from Trametes villosa. Factors of interest for the three-hour enzymatic treatment were pH and laccase concentration. Under optimum Fenton reaction conditions, 80% conversion of the initial benzene concentration was achieved, giving a mixture containing oxidative dimerization product (biphenyl) and hydroxylation products (phenol, catechol, resorcinol, benzoquinone and hydroquinone). Enzymatic removal of biphenyl and benzoquinone was not possible but 2.5 U/mL laccase was successful in removal of the rest of the phenolic

Soybean peroxidase-catalyzed removal of an aromatic thiol, 2-mercaptobenzothiazole, from water.

This paper demonstrates, for the first time, the capability of soybean peroxidase (SBP), an enzyme, for catalyzing the removal of an aromatic thiol, 2-mercaptobenzothiazole (MBT), from aqueous solution. The optimum pH for enzymatic conversion of MBT in aqueous buffer was found to be in the range 6.0 to 9.0. The optimum hydrogen peroxide (H2O2): MBT stoichiometry was 0.6. In terms of standard units (U) of catalytic activity, the minimum SBP concentration required for 95% conversion of 1.0 mM MBT in 3 hours was found to be 0.9 U/mL. The presence of polyethylene glycol at 50 mg/L can reduce the enzyme concentration required for the same conversion by 3-fold. It is proposed that these findings should be the basis for viable and cost-effective treatment of MBT in industrial wastewater and/or process water.

Laccase-catalyzed removal of diphenylamine from synthetic wastewater.

The priority pollutant lists of both the U.S. Environmental Protection Agency (U.S. EPA) and the European Union (EU) include diphenylamine (DPA), a contaminant found in wastewater of various industries. This work demonstrates the potential of using enzymatic treatment to remove DPA from buffered synthetic wastewater. This treatment method includes oxidative polymerization of DPA using laccase from Trametes villosa, followed by removal of those polymers via adsorptive micellar flocculation (AMF) using sodium lauryl sulfate (SDS) and alum. Researchers investigated the effects of pH, laccase concentration, molecular mass, and concentration of polyethylene glycol (PEG) in continuously stirred batch reactors to achieve 95% substrate conversion in three hours. Treatment of 0.19 mM DPA was best at pH 7 and an enzyme concentration from 0.0025 to 0.0075 standard activity unit/mL. Except for PEG400 optimum enzyme and PEG concentrations decreased with an increase in PEG molecular mass. Optimum AMF conditions were pH 3.0 to 6.5, 200 mg/L of SDS, and 150 mg/L of alum.

Laccase-catalyzed removal of 2,4-dimethylphenol from synthetic wastewater: effect of polyethylene glycol and dissolved oxygen.

The potential use of laccase (SP-504) in an advanced oxidation-based treatment technology to remove 2,4-dimethylphenol (DMP) from water was investigated with and without the additive, polyethylene glycol (PEG). The DMP concentration was varied between 1.0 and 5.0 mM. The optimization of pH and enzyme concentration in the presence and absence of PEG was carried out. All experiments were carried out in continuously stirred reactors for 3h at room temperature. The reaction was initiated by adding enzyme to the reaction mixture. For more than 95% removal of DMP, the presence of PEG reduced the inactivation of enzyme so that the required enzyme concentrations were reduced by about 2-fold compared to the same reactions in the absence of PEG. Finally, the PEG concentrations were optimized to obtain the minimum dose required. For higher substrate concentrations, the availability of oxygen was insufficient in achieving 95% or more removal. Therefore, the effect of increasing dissolved oxygen at higher substrate concentration was investigated. The laccase studied was capable of efficiently removing DMP at very low enzyme concentrations and hence shows great potential for cost-effective industrial applications.

Inactivation of enzyme laccase and role of cosubstrate oxygen in enzymatic removal of phenol from water.

Research was conducted to evaluate the potential use of laccase and its susceptibility to inactivation in an alternative enzyme-based treatment technology to remove parent phenol from buffered distilled water. Enzymatic oxidative polymerization of phenol with laccase was carried out in continuously stirred batch reactors. The reaction products were insoluble polymers, which precipitated out of the solution once their solubility limits were exceeded. The findings demonstrated that the polymeric products had significant effects on enzyme activity consumption and subsequent phenol removal. Enzyme species present in the reaction vessel were classified into enzyme remaining in the solution (type 1) and enzyme adhering to the precipitate polymers (type 2). Type 1 enzyme was more efficient in removal of phenol from solution compared with type 2. Subsequent filtration enhanced the phenol removal by removing type 2 enzyme adhering to the polymer particles and decelerating enzyme inactivation. The study also investigated the effects of available dissolved oxygen, provided through aeration and hydrogen peroxide addition, on phenol removal. Aeration and hydrogen peroxide addition increased the dissolved oxygen concentration, but had no effect on the progress curve for phenol removal.

Removal of dinitrotoluenes from water via reduction with iron and peroxidase-catalyzed oxidative polymerization: a comparison between Arthromyces ramosus peroxidase and soybean peroxidase.

A two-step process for the removal of dinitrotoluene from water is presented: zero-valent iron reduction is coupled with peroxidase-catalyzed polymerization of the resulting diaminotoluenes (DAT). The effect of pH was examined in the reduction step: at pH 6 the reaction occurred much more rapidly than at pH 8. In the second step, optimal pH and substrate ratio, minimal enzyme concentration and effect of polyethylene glycol (PEG) as an additive for greater than 95% conversion of DAT, over a 3h reaction period were determined using high performance liquid chromatography. Two enzymes were investigated and compared: Arthromyces ramosus peroxidase (ARP) and soybean peroxidase (SBP). The optimal pH values were 5.4 and 5.2 for ARP and SBP, respectively, but SBP was more resistant to mild acid whereas ARP was more stable in neutral solutions. SBP was found to have a greater hydrogen peroxide demand (optimal peroxide/DAT molar ratio for SBP: 2.0 and 3.0 for 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT), respectively; for ARP: 1.5 and 2.75 for 2,4-DAT and 2,6-DAT, respectively) but required significantly less enzyme (0.01 and 0.1 U ml(-1) for 2,4-DAT and 2,6-DAT, respectively) to convert the DAT than ARP (0.4 and 1.5 U ml(-1) for 2,4-DAT and 2,6-DAT, respectively). PEG was shown to have no effect upon the degree of substrate conversion for either enzyme.

Municipal landfill leachate treatment for metal removal using water hyacinth in a floating aquatic system.

Experiments were carried out to investigate the ability of water hyacinth (Eichhornia crassipes) to remove five heavy metals (cadmium, chromium, copper, nickel, and lead) commonly found in leachate. All experiments were conducted in batch reactors in a greenhouse. It was found that living biomass of water hyacinth was a good accumulator for copper, chromium, and cadmium. The plants accumulated copper, chromium, and cadmium up to 0.96, 0.83, and 0.50%, respectively, of their dry root mass. However, lead and nickel were poorly accumulated in water hyacinth. Also, nonliving biomass of water hyacinth dry roots showed ability to accumulate all metals, except Cr(VI), which was added in anionic form. The highest total metal sorption by nonliving dry water hyacinth roots was found to take place at pH 6.4. The current research demonstrates the potential of using water hyacinth for the treatment of landfill leachate containing heavy metals.

Laccase-catalyzed removal of bisphenol-A from water: protective effect of PEG on enzyme activity.

The feasibility of using the enzyme laccase to treat synthetic wastewater containing bisphenol-A (BPA) was examined. Optimization of pH, laccase concentration, polyethylene glycol (PEG) as an additive for >95% conversion and precipitation of BPA over 3 h of reaction period was determined through colorimetric assay and HPLC. PEG reduced enzyme inactivation, allowing a 5.2-fold reduction in the amount of laccase required for >95% removal of BPA in the range of 0.1-1 mM over 3 h. The fate of PEG after the reaction was also monitored. Linear relationships were found between the concentration of BPA (0.1-1 mM) and the optimum concentrations of laccase and PEG. Little PEG remained in the solution when up to 75 mg/L of PEG was used to treat 0.5 mM BPA. Beyond this level, PEG concentration increased linearly in the supernatant. It is inferred that an interaction between PEG and the polymeric products resulted in the protection of laccase.

Growth of water hyacinth in municipal landfill leachate with different pH.

Batch experiments were conducted to investigate the effect of municipal landfill leachate pH on the growth of water hyacinth (Eichhornia crassipes). These experiments were carried out in a green house environment on leachate samples collected from Essex-Windsor Regional Landfill, Windsor, Ontario, Canada. It was found that water hyacinth plants survived in a pH range of 4.0 to 8.0. Both alkaline pH (above 8.0) and highly acidic pH (below 4.0) had inhibitory effect on the growth of plants. The pH range, for optimum growth of the water hyacinth plants was found to be 5.8 to 6.0. At optimum growth, water hyacinth had an average mean relative growth rate of 0.043 d-1. It was found that nitrogen compounds underwent different transformations depending on the pH of leachate. Plant uptake, nitrification and volatilization were among these transformations.

Enzyme-catalyzed removal of phenol from refinery wastewater: feasibility studies.

Phenols are present in petroleum refining wastewater. An enzymatic method for removing phenols from industrial aqueous effluent has been developed in the past several years. In this method, a peroxidase enzyme catalyzes the oxidation of phenol by hydrogen peroxide generation of phenoxyl radicals. These radicals diffuse from the active center of the enzyme into solution and react nonenzymatically to eventually form higher oligomers and polymers, which can be removed from wastewater by conventional coagulation and sedimentation or filtration. In this study, Arthromyces ramosus peroxidase (ARP) was applied to treat a petroleum refining wastewater containing 2 mM (188 mg/L) phenol in a batch and continuous-flow system. The latter consisted of a plug-flow reactor (PFR) where the reaction took place between phenol and hydrogen peroxide catalyzed by the enzyme in the presence of polyethylene glycol (PEG). A flocculation tank followed the PFR where alum and sodium hydroxide were added and then the polymers formed were settled in a sedimentation tank and removed from the system. Most (95 to 99%) of the phenol was removed by the same dose of ARP required for the treatment of synthetic wastewater containing an equal amount of phenol. Polyethylene glycol, as an additive, reduced enzyme inactivation and consequently reduced the enzyme dose and the cost of the treatment process. Step feeding of hydrogen peroxide was not effective in reducing the enzyme requirement. A significant removal of chemical oxygen demand was achieved when using PEG to reduce the enzyme dose.

A continuous system for Fe0 reduction of nitrobenzene in synthetic wastewater.

Nitrobenzene is a major environmental pollutant, and its degradation is difficultto achieve. Hence, a chemical reduction pretreatment is sought in this research, before the resulting aniline can be treated by enzyme-mediated oxidative polymerization. Zerovalent iron (Fe0) has been successfully employed to reduce nitrobenzene to aniline in synthetic wastewater in both batch and continuous flow reactors. The concentration of nitrobenzene studied was thatwhich would be present in industrial wastewater streams (millimolar, 123 ppm), a concentration range considerably higher than those studied previously with groundwater by other researchers. Anaerobic conditions were maintained in the reactors by including Na2SO3 as an oxygen scavenger in the presence of CoCl2.6H2O, which acted as a catalyst. Batch reactors exhibited adsorption of aniline on the Fe0, which could be described by a langmuir isotherm. A 200 g Fe0 (particle size: 1-2 mm) bed completely converted 1 mM of nitrobenzene flowing upward for about 600 pore-volumes before experiencing flow reduction due to clogging due to corrosion products. Green-black precipitates (Fe0 corrosion products) were formed at the influent end of the column which were identified as maghemite.

Extraction of elemental sulfur from an aqueous suspension for analysis by high-performance liquid chromatography.

A bioreactor is being developed that produces elemental sulfur suspended in aqueous bioreactor contents. The concentration of elemental sulfur must be measured explicitly in order to study the efficiency of the conversion of sulfide to elemental sulfur. Extracting the sample with ethanol gave erroneous results when sulfide was present in solution. The extraction of aqueous elemental sulfur into petroleum ether prior to colorimetric determination was tested. When the aqueous matrix was simply deionized water, the extraction was poor. The development of a method of extraction of the sulfur into chloroform prior to quantification by high-performance liquid chromatography is described. The efficiency of the extraction was found to be greater than 90% in all matrixes tested and linear for aqueous elemental sulfur concentrations up to 200 mg/L.

Enzymatic removal of selected aromatic contaminants from wastewater by a fungal peroxidase from Coprinus macrorhizus in batch reactors.

The use of enzymes such as horseradish peroxidase (HRP) for degrading or removing toxic organics from synthetic wastewater has been demonstrated previously. Potential alternatives to HRP are other peroxidases, various ligninases, haloperoxidases and laccases. Results of this study indicate that a fungal peroxidase from Coprinus macrorhizus (CMP) has the capability to catalyze the same reactions as HRP. Similarly, in batch reactors the trend and removal efficiency of aromatic compounds by CMP from synthetic wastewater depend on the nature of the compound.

Inactivation of horseradish peroxidase by phenol and hydrogen peroxide: a kinetic investigation.

Inactivation of horseradish peroxidase (HRP) was examined in the presence of hydrogen peroxide alone and in the presence of hydrogen peroxide plus phenol. HRP is inactivated upon exposure to hydrogen peroxide (H2O2) by the combination of two possible pathways, dependent upon hydrogen peroxide concentration. At low H2O2 concentrations (below 1.0 mM in the absence of phenol), inactivation is predominantly reversible, resulting from the formation and accumulation of catalytically inert intermediate compound III. As H2O2 concentrations increase, an irreversible mechanism-based inactivation process becomes predominant. The overall inactivation comprised of both processes exhibits a second-order inactivation rate constant (kapp) of 0.023 +/- 0.005 M-1 s-1 at pH 7.4 and 25 degrees C. In the presence of both hydrogen peroxide fixed at 0.5 mM and phenol, HRP was inactivated in an irreversible, time- and phenol concentration-dependent process, also mechanism-based, with a kapp of 0.019 +/- 0.004 M-1 s-1.