Jean Marc Bollag

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

Cross-linkage between anilines and phenolic humus constituents

W The pesticide degradation intermediates 4-chloroaniline, 3,4-dichloroaniline, and 2,6-diethylaniline were examined for their ability to react with various phenolic humus constituents in the presence of a fungal phenol oxidase. An initial indication of a cross-coupling reaction was obtained by color formation. Although anilines alone were not transformed by a phenol oxidase isolated from the fungus Rhizoctonia praticola, they readily cross-linked with phenolic acids or their enzymatic products to form hybrids in the presence of the enzyme. A number of hybrid dimers, trimers, and tetramers were isolated and determined by mass spectrometric analysis. The identities of three hybrid dimers formed from syringic acid coupled with 4-chloroaniline, 3,4-dichloroaniline, or 2,g-diethylaniline and a hybrid tetramer from protocatechuic acid coupled with 2,6-diethylaniline were determined. The formation of the hybrid dimer N-(2,6-diethylphenyl)-2,6dimethoxy-p-benzoquinone imine from a cross-linkage between syringic acid and 2,6-diethylaniline was quantitatively evaluated.

Enzyme-mediated coupling of 3,4-dichloroaniline and ferulic acid: a model for pollutant binding to humic materials.

To investigate the incorporation of chloroanilines into humic substances, the reaction of ferulic acid with three different chloroanilines in the presence of a laccase of the fungus Rhizoctonia praticola was investigated. Under the reaction conditions used, chloroanilines alone were not transformed by the laccase, but in the presence of ferulic acid they formed cross-coupling products. The reactivity of anilines decreased with increasing chlorine substitution on the ring. Two dimeric compounds were isolated from the reaction assay of ferulic acid alone. The dimers were found to have phenylcoumaran and lactone structures

Aerobic versus anaerobic metabolism of halogenated anilines by aParacoccus sp.

AParacoccus sp. which transforms aniline and different halogen-substituted derivatives under aerobic and anaerobic conditions was isolated from the soil. In experiments with14C-ring-labeled 4-chloroaniline, approximately 60% of the radioactive material disappeared from the growth medium after incubation under anaerobiosis within 48 hr, but under aerobic conditions no decrease of radioactivity in the growth medium was observed, although 4-chloroaniline was completely metabolized. Acetylation appears to constitute, especially under aerobic conditions, a major transformation mechanism by the bacterium, since almost 50% of the acetylated compound could be detected and identified if aniline, 2-, 3-, and 4-chloroaniline served as substrate. The formation of different metabolites under aerobic and anaerobic conditions clearly indicates the existence of two separate pathways in the metabolism of aniline compounds depending on the oxygen status of the environment.

A column study of the biological mobilization and speciation of cadmium in soil

An acid sandy loam soil was used in a column study to determine the effect of microorganisms on Cd mobilization in soil and its speciation in column leachates. Non-sterile and sterile (gamma-irradiated) soil columns were initially treated with 90 mg of Cd (NO3)2, and approximately 20 mg of added Cd was retained in the soil. Soil columns were subsequently eluted with a sterile nutrient medium for a period of up to 38 days. Non-sterile and sterile soil columns leached with sterile deionized water served as unsupplemented controls. During a leaching period of 38 days, about 36% of the sorbed Cd in soil was mobilized in the presence of microorganisms and nutrients, while less than 16% of the Cd was released from the sterile and unsupplemented soil controls. To characterize the chemical form of the microbially mobilized Cd, the leachates were analyzed by gel filtration chromatography. In this study, 86% to 93% of the mobilized Cd was associated with the low molecular weight inorganic and organic fraction, while the remainder was eluted in the high molecular weight range. The low molecular weight Cd fractions were further examined by solid phase extraction (C18 Sep-Pak), and cation-exchange chromatography. From the various analyses, it was concluded that a significant amount of Cd existed as a hydrophilic organic complex, and the remainder as cationic Cd. Soil microorganisms were mostly responsible for the transformation of Cd in soil and its subsequent mobilization.

Immobilization of cadmium by microbial extracellular products

An axenic bacterial culture was isolated from Morrison loam soil (pH 6.4) and was capable of growth in the presence of CdO (175 μg/mL of Cd). During the first two days of incubation, growth of the culture was accompanied by a decrease in the concentration of soluble Cd. This effect was due to two phenomena: (1) a decrease in the solubility of CdO in medium conditioned by the microorganisms; and (2) to sorption of Cd to bacterial cells. The solubility of CdO in filter-sterilized spent medium was very low (4–6 μg/mL), yet the solubility was high (110–160 μg/mL) in autoclaved, filtered spent medium. Changes in the pH value of the medium were not responsible for this effect. Incubation of spent medium in the presence of increasing concentrations of Cd as Cd(NO3)2 resulted in a decrease in the metal concentration of the liquid, due to the formation of a precipitate in quantities proportional to the amount of added Cd. Similar precipitate formation was observed when 100 μg/mL of Hg, Cu, or Zn were added but not when 100 μg/mL of Mg was introduced. The compound(s) responsible for the precipitate formation was not dialyzable from tubing with a pore size of 12,000–14,000 and was not retained by a C18 Sep-Pak cartridge. Dialysis of a suspension of the precipitate against water (pH 3.0) resulted in partial dissolution of the complex. Approximately 50% of the bound Cd was released and this was accompanied by the release of a protein of molecular weight of 42,600 daltons. The results suggest that this microbial protein forms a water insoluble precipitate with Cd and therefore plays a major role in decreasing the concentration of soluble Cd during microbial growth.

A model for enzymatic binding of pollutants in the soil.

Abstract Pollutants released into the environment may be bound to humic acid through abiotic or biologic processes whereby the formation of bound residues usually results in detoxification of the pollutant. Therefore, enhancing the binding of xenobiotic chemicals to humic material can serve as a means to reduce toxicity as well as migration of toxic compounds in the environment. Complex formation can occur by an oxidative coupling reaction leading to oligomeric and polymeric products. We intensively investigated the effect of phenoloxidases (peroxidases, tyrosinases, and laccases) on the binding of substituted phenols and aromatic amines to humus monomers as well as to humic substances. Based on a large number of isolated and identified cross-coupling products, we are able to elucidate the sites and mechanisms of binding and also to determine the efficiency of some coupling reactions. Copolymerization largely depends on the chemical reactivity of the substrates involved. Certain phenolic humus constituent...

Effect of the physiological state of microbial cells on cadmium sorption

Live and dead cells of several strains of bacteria as well as different microbial populations isolated from natural environments were tested for their ability to sorb cadmium (Cd) from a liquid medium (0.05M phosphate buffer, pH 7.2) containing 10 μg Cd/ml. The bacterial cultures were grown in a nutrient solution without Cd, harvested, and then suspended in the buffer solution for the uptake assays. The amount of Cd sorbed by dead cells was usually higher than by live cells. Killing of bacteria by chemicals (ethanol, formaldehyde, or propylene oxide) or physical (γ-radiation) means had a varying effect on Cd uptake by dead cells. Various bacterial strains showed different degrees of resistance to Cd and different uptake levels of this heavy metal with no established correlation. Neither storage of the cells at 4°C for 48 hr after harvesting nor varying incubation times of the bacteria before the Cd uptake experiments had any influence on the amount of Cd sorbed.

Aerobic TNT reduction via 2-hydroxylamino-4,6-dinitrotoluene by Pseudomonas aeruginosa strain MX isolated from munitions-contaminated soil

Bioremediation of munitions-contaminated soil requires effective transformation and detoxification of high concentrations of 2,4,6-trinitrotoluene (TNT). Pseudomonas aeruginosa strain MX, isolated from munitions-contaminated soil, aerobically transformed TNT (100 mg/L) in culture medium within 15 h, causing transient accumulation of hydroxylaminodinitrotoluenes (HADNTs). The predominance of 2-hydroxylamino-4,6-dinitrotoluene (2HADNT), as well as 2-amino-4,6-dinitrotoluene (2ADNT) and 4,4′,6,6′ -tetranitro-2,2′ -azoxytoluene (2,2′AZT), indicated preferential reduction of the TNT ortho nitro group. While only 12% of the TNT was transformed to 2ADNT, up to 65% was transformed to tetranitroazoxytoluenes (AZTs), which accumulated as a precipitate. The precipitate was formed by microscopic particles adhering to bacterial cells, which subsequently formed clusters containing lysed cells. Toxicity toward bacteria was primarily attributed to 2ADNT, because pure AZTs preincubated with sterile medium had little effect on the strain. While the culture medium containing TNT exhibited toxicity toward corn (Zea mays L.) and witchgrass (Panicum capillare L.), little phytotoxicity was observed after incubating with P. aeruginosa strain MX for 4 d. Strong binding of HADNTs to soil and low AZT bioavailability may further promote the detoxification of TNT in soil.