James A. Nicell

Potential Applications of Enzymes in Waste Treatment

The implementation of increasingly stringent standards for the dis- charge of wastes into the environment has necessitated the need for the develop- ment of alternative waste treatment processes. A review of research directed toward developing enzymatic treatment systems for solid, liquid and hazardous wastes is presented. A large number of enzymes from a variety of di†erent plants and microorganisms have been reported to play an important role in an array of waste treatment applications. Enzymes can act on speci-c recalcitrant pollutants to remove them by precipitation or transformation to other products. They also can change the characteristics of a given waste to render it more amenable to treatment or aid in converting waste material to value-added products. Before the full potential of enzymes may be realized, it is recommended that a number of issues be addressed in future research endeavors including the identi-cation and characterization of reaction by-products, the disposal of reaction products and reduction of the cost of enzymatic treatment.

A model of peroxidase activity with inhibition by hydrogen peroxide

The rate of color formation in an activity assay consisting of phenol and hydrogen peroxide as substrates and 4-aminoantipyrine as chromogen is significantly influenced by hydrogen peroxide concentration due to its inhibitory effect on catalytic activity. A steady-state kinetic model describing the dependence of peroxidase activity on hydrogen peroxide concentration is presented. The model was tested for its application to soybean peroxidase (SBP) and horseradish peroxidase (HRP) reactions based on experimental data which were measured using simple spectrophotometric techniques. The model successfully describes the dependence of enzyme activity for SBP and HRP over a wide range of hydrogen peroxide concentrations. Model parameters may be used to compare the rate of substrate utilization for different peroxidases as well as their susceptibility to compound III formation. The model indicates that SBP tends to form more compound III and is catalytically slower than HRP during the oxidation of phenol.

Removal of phenols from a foundry wastewater using horseradish peroxidase

Horseradish peroxidase (HRP) catalyses the oxidation of phenols by hydrogen peroxide resulting in the formation of water-insoluble polymers which can be separated by coagulation and sedimentation. The feasibility of the enzyme process to treat a foundry wastewater containing 3.5 mM of total phenols (330 mg/l as phenol) was examined. Two enzyme stocks of different purities were used but total phenols removal was independent of enzyme purity. For both stocks, 97 to 99% of the phenolic contaminants were removed, despite the presence of other contaminants such as organic compounds and iron in the waste matrix. The quantity of HRP required for this degree of treatment was in the same range as for the treatment of a synthetic wastewater containing an equal amount of pure phenol. Polyethylene glycol, a chemical additive, reduced enzyme inactivation, allowing a 22-fold reduction in the amount of HRP required for 99% removal of phenols from the foundry waste. Residual chemical oxygen demands (COD) varied depending on the enzyme source. The high purity HRP achieved more than 65% removal of COD, but due to a high concentration of other organic matter present in the low purity HRP, no reduction in COD was achieved with this enzyme source. A comparison was made between enzyme treatment and oxidation using Fentons reagent. Enzyme cost must be significantly reduced in order to make the enzyme treatment process economically competitive.

Characterization of soybean peroxidase for the treatment of aqueous phenols

The application of soybean peroxidase (SBP) to catalyze the polymerization and precipitation of aqueous phenols by hydrogen peroxide is potentially promising because this peroxidase is less expensive than horseradish peroxidase (HRP), which has been the focus of most wastewater research. SBP can act on a broad range of compounds and retains its catalytic ability over wide ranges of temperature and pH. Activity was optimal at pH 6.4, with significant activity observed between pH 3 and 9. SBP was very stable at 25°C at neutral and alkaline conditions but experienced rapid inactivation below pH 3. SBP underwent biphasic inactivation by hydrogen peroxide in the absence of a reductant substrate. SBP was most effective when used to treat phenolic solutions between pH 6 and 9. In comparison with HRP, the activity of SBP was only slightly more sensitive to pH, was more stable at elevated temperatures, and was less susceptible to permanent inactivation by hydrogen peroxide. However, SBP was catalytically slower than HRP and a larger molar quantity of SBP was usually required to remove a given quantity of phenolic substrate.

Detoxification of phenolic solutions with horseradish peroxidase and hydrogen peroxide

Phenolic solutions were treated with hydrogen peroxide and horseradish peroxidase (HRP) resulting in more than 95% removal of phenols within 3 h. Toxic compounds were formed during the treatment of aqueous solutions of phenol, 2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol and 2-methylphenol. However, the toxicities of HRP-treated solutions decreased within 21 h after the completion of the enzymatic reaction, except in the case of 2-methylphenol. The process of detoxification was significantly accelerated upon the addition of hydrogen peroxide to the dephenolized solutions. Solutions that were treated in the presence of chitosan exhibited lower toxicities than solutions treated in its absence if they were allowed to incubate for an extended period of time. Treatment in the presence of polyethylene glycol resulted in significantly higher toxicities. The toxicity of treated solutions was dependent on the addition mode of HRP and hydrogen peroxide. Treated solutions were also completely detoxified following illumination with UV light.

Model development for horseradish peroxidase catalyzed removal of aqueous phenol.

Once activated by hydrogen peroxide, horseradish peroxidase (HRP) catalyzes the oxidation of aqueous aromatic compounds to produce high molecular weight polymers of low solubility. A pseudo steady-state kinetic model of the HRP-hydrogen peroxide-aromatic compound system was modified to incorporate enzyme inactivation mechanisms in order to improve its predictive ability. The kinetic constants of the model were calibrated using a series of experimental data sets. The models ability to predict the time-dependent removal of phenol within the range of 0.5-6 mM from a batch reactor was validated. The model accounts for permanent losses of enzyme activity through inactivation by free radicals as well as interaction with end-product polymers as they form.

Treatment of aqueous phenol with soybean peroxidase in the presence of polyethylene glycol

Abstract Soybean peroxidase (SBP) catalyzes the oxidation and polymerization of aromatic compounds in the presence of hydrogen peroxide. The polymerized products precipitate from solution, providing an alternative to conventional treatment methods. Studies were conducted to characterize the use of polyethylene glycol (PEG) as an additive to increase the active life of the enzyme. The effectiveness of PEG increased with its molecular weight, with maximum protection accomplished with PEG of molecular weight 35,000. Linear relationships were found between the quantity of phenol to be treated (1.0–10 mM) and the optimum doses of SBP and PEG required for greater than 95% removal. Observations suggest that it is the interaction between the PEG and the polymeric products that results in the protection of SBP. Following treatment, approximately 25% of the optimum PEG dose remained in the supernatant.

REACTOR DEVELOPMENT FOR PEROXIDASE CATALYZED POLYMERIZATION AND PRECIPITATION OF PHENOLS FROM WASTEWATER

Abstract Horseradish peroxidase enzyme catalyzes the oxidation of toxic aromatic compounds, especially phenols and aromatic amines, in the presence of hydrogen peroxide. The reaction products polymerize to form high molecular weight materials which readily precipitate from solution; hence, providing a means for the treatment of wastewaters which contain aromatic compounds. The catalytic lifetime of horseradish peroxidase enzyme can be extended by optimizing treatment conditions such as pH and temperature and by maintaining a low instantaneous enzyme concentration in the reaction mixture. The enzyme catalyzed polymerization process was implemented in a continuous stirred tank reactor (CSTR) configuration because reactant and enzyme concentrations are lowered immediately upon entering the reactor causing a reduction in inactivation of HRP through free radical bonding and compound III formation. Catalytic turnovers achieved in single and multiple CSTRs in series were significantly higher than those observed in batch reactors when sufficient retention time was provided.

Characterization of tyrosinase for the treatment of aqueous phenols

Abstract Mushroom tyrosinase (polyphenol oxidase, EC 1.14.18.1) was investigated as an alternative to peroxidase enzymes for the catalytic removal of phenolic compounds from wastewaters. Maximum catalytic activity was observed at pH 7 and more than 50% of optimum activity was observed at pHs ranging between 5 and 8. Tyrosinase was unstable under acidic conditions and at elevated temperatures. The activation energy for thermal inactivation of tyrosinase at pH 7 was determined to be 1.85 kJ mol −1 using l -tyrosine as a substrate. Phenol was successfully transformed by tyrosinase over wide ranges of pH 5–8 and initial phenol concentration (0.5–10 mM, 47–940 mg/l). Several chlorinated phenols were also successfully transformed. Polyethylene glycol and chitosan did not protect tyrosinase from inactivation during the treatment of phenol; however, chitosan induced the precipitation of reaction products arising from phenol transformation.

Biodegradation of plasticizers by Rhodococcus rhodochrous.

Rhodococcus rhodochrous was grown in the presence of oneof three plasticizers: bis 2-ethylhexyl adipate (BEHA), dioctyl phthalate (DOP) ordioctyl terephthalate (DOTP). None of the plasticizers were degraded unless anothercarbon source, such as hexadecane, was also present. When R. rhodochrous was grownwith hexadecane as a co-substrate, BEHA was completely degraded and the DOP was degraded slightly. About half of the DOTP was degraded, if hexadecane were present.In all of these growth studies, the toxicity of the media, which was assessed usingthe Microtox assay, increased as the organism degraded the plasticizer. In each case, therewas an accumulation of one or two intermediates in the growth medium as the toxicityincreased. One of these was identified as 2-ethylhexanoic acid and it was observed forall three plasticizers. Its concentration increased until degradation of the plasticizershad stopped and it was always present at the end of the fermentation. The other intermediatewas identified as 2-ethylhexanol and this was only observed forgrowth in the presence of BEHA. The alcohol was observed early in the growth studies with BEHA and haddisappeared by the end of the experiment. Both the 2-ethylhexanol and 2-ethylhexanoicacid were shown to be toxic and their presence explained the increase of toxicity asthe fermentations proceeded. The appearance of these intermediates was consistent with similar degradation mechanisms for all three plasticizers involving hydrolysisof the ester bonds followed by oxidation of the released alcohol.