Jerzy Dec

Detoxification of substituted phenols by oxidoreductive enzymes through polymerization reactions

Laccases from the fungiRhizoctonia praticola andTrametes versicolor as well as horseradish peroxidase and tyrosinase were evaluated for their ability to polymerize phenolic contaminants. The removal of phenols through polymerization depended on the chemical structure and concentration of the substrate, pH of the reaction mixture, activity of the enzyme, length of incubation, and temperature. The enzymes retained their activity throughout a broad range of pH (pH 3.0 to 10) and temperature (5 to 55°C). The removal of halogenated phenols decreased with increasing number of chlorines and increasing molecular weight of the substituent. Laccases fromR. praticola andT. versicolor removed 2,4-dichlorophenol at initial concentrations of up to 1,600 mg/L. The amount of the substrate removed increased with increasing enzyme activity. The precipitates formed during polymerization of 2,4-dichlorophenol constituted a mixture of oligomers with average molecular weights of up to 800 for the fraction soluble in dioxane. Mass spectra revealed the loss of chlorine atoms during enzymatic polymerization. The release of chloride ions into solution during polymerization amounted to up to 20% of the chlorine initially associated with the 2,4-dichlorophenol molecule. Dechlorination contributes to the overall detoxification effect which results from enzymatic polymerization.

Dehalogenation of chlorinated phenols during oxidative coupling

Dehalogenation of chlorophenols by oxidoreductive enzymes is well-documented, but the cause of this phenomenon has not yet been determined. In this study, release ofchloride ions was observed during two oxidative coupling processes, polymerization of chlorinated phenols and their binding to humic acid. The reactions were catalyzed by two oxidoreductases and an inorganic catalyst (birnessite). The dehalogenation patterns indicated that release of chloride ions is caused by the free-radical reaction of oxidative coupling and takes place when the unpaired electron of a free radical is located at a chlorine-substituted aromatic carbon. These findings have helped to elucidate various aspects of oxidative coupling during polymerization and binding processes

Removal of Odors from Swine Wastewater by Using Microbial Fuel Cells

ABSTRACT A single-chamber microbial fuel cell (MFC) was used to reduce 10 chemicals associated with odors by 99.76% (from 422 ± 23 μg/ml) and three volatile organic acids (acetate, butyrate, and propionate) by >99%. The MFC produced a maximum of 228 mW/m2 and removed 84% of the organic matter in 260 h. MFCs were therefore effective at both treatment and electricity generation.

Determination of covalent and noncovalent binding interactions between xenobiotic chemicals and soil

Knowledge of the fate of xenobiotics in terrestrial systems is an important aspect of soil science. This paper reviews experimental approaches that have enhanced our understanding of binding interactions between xenobiotic chemicals and soil. First attempts to evaluate the nature of binding focused on the identification of covalently bound or physically sequestered chemicals upon their removal from the soil matrix by alkaline or acid hydrolysis and other release techniques (e.g., high-temperature distillation, supercritical fluid extraction or microwave extraction). The covalent nature of bonds formed was confirmed by model studies in which xenobiotic chemicals, such as phenols or anilines, were allowed to interact with monomeric constituents of humus (e.g., syringic acid or guaiacol). Further studies involved 13 C or 15 N NMR analysis of 13 C- or 15 N-labeled xenobiotics that were bound to natural humic acid or soil. The resolution of NMR spectra was greatly improved by silylation ofthe soil samples and application of 13 C-depleted humic materials. NMR spectroscopy in combination with silylation was also instrumental in the evaluation of physically sequestered chemicals. A coherent theory of sequestration (dual-mode sorption model) was developed based on adsorption isotherms obtained in experiments involving long contact times between xenobiotics and soil. The future of research on binding appears to depend largely on NMR spectroscopy and further progress in our knowledge of humus.

Release of substituents from phenolic compounds during oxidative coupling reactions

Phenolic compounds originating from plant residue decomposition or microbial metabolism form humic-like polymers during oxidative coupling reactions mediated by various phenoloxidases or metal oxides. Xenobiotic phenols participating in these reactions undergo either polymerization or binding to soil organic matter. Another effect of oxidative coupling is dehalogenation, decarboxylation or demethoxylation of the substrates. To investigate these phenomena, several naturally occurring and xenobiotic phenols were incubated with various phenoloxidases (peroxidase, laccase, tyrosinase) or with birnessite (delta-MnO(2)), and monitored for chloride release, CO(2) evolution, and methanol or methane production. The release of chloride ions during polymerization and binding ranged between 0.2% and 41.4%. Using the test compounds labeled with 14C in three different locations (carboxyl group, aromatic ring, or aliphatic chain), it was demonstrated that 14CO(2) evolution was mainly associated with the release of carboxyl groups (17.8-54.8% of the initial radioactivity). Little mineralization of 14C-labeled aromatic rings or aliphatic carbons occurred in catechol, ferulic or p-coumaric acids (0.1-0.7%). Demethoxylation ranged from 0.5% to 13.9% for 2,6-dimethoxyphenol and syringic acid, respectively. Methylphenols showed no demethylation. In conclusion, dehalogenation, decarboxylation and demethoxylation of phenolic substrates appear to be controlled by a common mechanism, in which various substituents are released if they are attached to carbon atoms involved in coupling. Electron-withdrawing substituents, such as -COOH and -Cl, are more susceptible to release than electron-donating ones, such as -OCH(3) and -CH(3). The release of organic substituents during polymerization and binding of phenols may add to CO(2) production in soil.

Effect of various factors on dehalogenation of chlorinated phenols and anilines during oxidative coupling

According to previous research, dehalogenation of chlorinated phenols in the presence of horseradish peroxidase or Trametes versicolor laccase is due to the free-radical mechanism of an oxidative coupling reaction. This study demonstrated that short reaction time, limited amount of catalyst, or unfavorable pH conditions may result in an insufficient generation of free radicals and a reduction in dehalogenation of chlorinated phenols. The free-radical mechanism of chloride release was also found to be valid for peroxidase-mediated polymerization of chlorinated anilines. In the case of a tyrosinase-mediated polymerization of chlorophenols, chloride ions were apparently removed from the formed o-quinones during nucleophilic attack by phenoxide ions. The results obtained strongly support the hypothesis that dehalogenation is strictly connected to oxidative coupling and cannot be enhanced independently by adjustment of the reaction conditions.

Enhanced enzymatic removal of chlorophenols in the presence of co-substrates

Abstract The effect of reactive co-substrates such as guaiacol and 2,6-dimethoxyphenol on the removal of chlorinated phenols by horseradish peroxidase (HRP) and a laccase from the fungus Trametes versicolor was investigated. Addition of 50 mM guaiacol enhanced the precipitation of 4-chlorophenol, 2,4-dichlorophenol, and 2,4,5-trichlorophenol with peroxidase by 12, 32 and 65%, respectively, and with laccase by 20, 32 and 80%, respectively. Addition of 10 mM 2,6-dimethoxyphenol enhanced the precipitation of 2,4,5-trichlorophenol by 90% with peroxidase and by 98% with laccase. Products from the reaction of 2,6-dimethoxyphenol and peroxidase were filtered to exclude compounds of a molecular weight greater than 500. Incubation of the resulting enzyme-free filtrate with a solution of unreacted 2,4,5-trichlorophenol caused precipitation and a 72% removal of the 2,4,5-trichlorophenol. Chlorophenol precipitation in the presence of co-substrates may be a useful strategy for improving the efficiency of enzymatic decontamination methods, particularly in the case of heterogenous pollution.

Transformation of the fungicide cyprodinil by a laccase of Trametes villosa in the presence of phenolic mediators and humic acid

Xenobiotic chemicals can be transformed or covalently bound to humic materials by oxidoreductive enzymes present in terrestrial systems. Chemicals that are not substrates for oxidoreductive enzymes may undergo transformation in the presence of certain reactive compounds, which are often referred to as mediators. In this study, cyprodinil, a broad-spectrum fungicide, did not show any transformation when incubated alone with a laccase from Trametes villosa. It was transformed to a significant extent, however, when a mediator was present. All of the 13 tested mediators belonged to the group of naturally occurring phenols. With some exceptions (2,6-dimethoxyphenol, syringic acid, and ferulic acid), phenols substituted with one or two methoxy groups were very effective mediators. In experiments with 14C-labeled cyprodinil, the radioactive label was largely associated with brown transformation products that precipitated out of the aqueous solution. As determined by mass spectrometry, the products were mixed oligomers resulting from cross-coupling between cyprodinil and a mediator. The addition of large amounts of humic acid (HA) (400 mg/L) to the reaction mixtures involving the most effective mediators reduced cyprodinil transformation (42.6-68.6%) by 12-48%, probably due to an inhibitory effect. The inhibition decreased with decreasing concentration of HA. The addition of HA (400 mg/L) to the reaction mixtures involving the least effective mediators or no mediators (control) enhanced cyprodinil transformation (0.3-17.6%) by 2.9-17.1%, probably as a result of binding to HA.

Formation Mechanisms of Complex Organic Structures in Soil Habitats

Publisher Summary This chapter discusses the formation of complex organic structures in soil habitats. The purpose of the chapter is to review the existing knowledge about the synthetic processes previously outlined and to discuss their origins, importance, and practical implications. Chemoheterotrophs are the only organisms that can perform the two critical functions necessary to the humification process: firstly, an active role of transforming raw materials into humus, and secondly a passive one of serving as a source of biomass after death. Adsorption mechanisms constitute another important type of synthetic interaction responsible for binding of xenobiotics to soil organic matter. Xenobiotics retained through adsorption are considered reversibly bound, because they can be desorbed through extraction with organic solvents. The synthetic reactions discussed in this chapter are essential to the constant replenishment of humus. Another significant benefit of these reactions is the neutralization of certain natural soil organic substances, such as benzoic acid, vanillin, and other lignin decomposition products, which have been proved to be toxic to plants.

DECARBOXYLATION AND DEMETHOXYLATION OF NATURALLY OCCURRING PHENOLS DURING COUPLING REACTIONS AND POLYMERIZATION

Phenolic compounds originating from plant residue decomposition or microbial metabolism form humic-like polymers in the presence of various phenoloxidases or metal oxides. Enzyme-mediated reactions were reported to result in the decarboxylation or demethoxylation of substrate molecules; decarboxylation was also observed with metal oxides. To obtain more information on these phenomena, several humic precursors were incubated with various phenoloxidases (peroxidase, laccase, tyrosinase) or birnessite (δ-MnO2) and monitored for CO2 evolution and methanol production. Additionally, some reaction mixtures were analyzed for methane evolution. By using the test compounds labeled with 14C in three different locations (carboxyl group, aromatic, or aliphatic chain), we demonstrated that 14CO2 evolution (ranging from 4.6 to 63.5% of the initial radioactivity) was mainly associated with the release of carboxyl groups. Minimal mineralization of 14C-labeled aromatic rings or aliphatic carbons occurred in ferulic or p-coumaric acids (0–5.6%). Demethoxylation ranged from 0.5 to 13.9% for 2,6-dimethoxyphenol and syringic acid, respectively. The methyl groups in 2-, 3-, and 4-methylphenol resisted release, as indicated by the lack of methane or methanol production. In previous studies, chlorophenols incubated with various phenoloxidases or birnessite were subject to dehalogenation. It appears that dehalogenation, decarboxylation, and demethoxylation of phenolic substrates are controlled by a common mechanism, in which various substituents are released if they are attached to carbon atoms involved in coupling. According to the experimental data, electron-withdrawing substituents, such as –COOH and –Cl, are more susceptible to release than electron-donating ones, such as –OCH3 and –CH3. The release of organic substituents during polymerization of humic precursors may add to CO2 production in soil.