Biodegradation of endocrine-disrupting phenolic compounds using laccase followed by activated sludge treatment

Endocrine-disrupting phenolic compounds in the water were degraded by laccase fromTrametes sp. followed by activated sludge treatment. The effect of temperature on the degradation of phenolic compounds and the production of organic compounds were investigated using endocrine-disrupting chemicals such as bisphenol A, 2,4-dichlorophenol, and diethyl phthalate. Bisphenol A and 2,4-dichlorophenol disappeared completely after the laccase treatment, but no disappearance of diethyl phthalate was observed. The Michaelis-Menten type equation was proposed to represent the degradation rate of bisphenol A by the lacasse under various temperatures. After the laccase treatment of endocrine-disrupting chemicals, the activated sludge treatment was attempted and it could convert about 85 and 75% of organic compounds produced from bisphenol A and 2,4-dichlorophenol into H₂O and CO₂, respectively.     

Cadmium removal and 2,4-dichlorophenol degradation by immobilized Phanerochaete chrysosporium loaded with nitrogen-doped TiO2 nanoparticles

Phanerochaete chrysosporium has been identified as an effective bioremediation agent for its biosorption and degradation ability. However, the applications of P. chrysosporium are limited owing to its long degradation time and low resistance to pollutants. In this research, nitrogen-doped TiO₂ nanoparticles were loaded on P. chrysosporium to improve the remediation capacity for pollutants. The removal efficiencies were maintained at a high level: 84.2 % for Cd(II) and 78.9 % for 2,4-dichlorophenol (2,4-DCP) in the wide pH range of 4.0 to 7.0 in 60 h. The removal capacity of immobilized P. chrysosporium loaded with nitrogen-doped TiO₂ nanoparticles (PTNs) was strongly affected by the initial Cd(II) and 2,4-DCP concentrations. The hyphae of PTNs became tight, and a large amount of crystals adhered to them after the reaction. Fourier transform infrared spectroscopy showed that carboxyl, amino, and hydroxyl groups on the surface of PTNs were responsible for the biosorption. In the degradation process, 2,4-DCP was broken down into o-chlorotoluene and 4-hexene-1-ol. These results showed that PTNs is promising for simultaneous removal of Cd(II) and 2,4-DCP from wastewater.     

Use of plant material for the decontamination of water polluted with phenols

Plant materials were found useful in the decontamination water polluted with phenolic contained in the plant tissue. The enzymes mediated oxidative coupling of the pollutants, followed by precipitation of the formed polymers from the aqueous phase. An industrial wastewater contaminated with 2,4-dichlorophenol (up to 850 ppm) and other chlorinated phenols was successfully treated using minced horseradish, potato, or white radish (amended with H(2)O(2)). Horseradish-mediated removal of 2,4-dichlorophenol from model solutions was comparable with that achieved using purified horseradish peroxidase. In addition, horseradish could be reused up to 30 times. Due to the apparent ease of application, the use of plat material may present a breakthrough in the enzyme treatment of contaminated water. (c) 1994 John Wiley & Sons, Inc.     

The Development of a Phytoremediation Technique for the Detoxification of Soils Contaminated with Phenolic Compounds Using Horseradish Peroxidase (Armoracia rusticana): Preliminary Results

The proposed phytoremediation technique is based on the successful exploitation and optimization of oxidative coupling, mediated by horseradish peroxidase. Susceptibility to degradation of a selection of phenolic compounds, in solution, by horseradish peroxidase appears to be structurally related and was found to be of the order 2,4-dichlorophenol (2,4-DCP) > 4-chlorophenol (4-CP) > 2-chlorophenol (2-CP). Only 1.89% of 2,4-DCP, at an initial concentration of 5 mM, remained unchanged at the end of the experiment. Reaction rates between purified horseradish peroxidase and 2,4-DCP were found to be extremely rapid with 74% of the substrate removed from solution during the first 30 s. Inhibition of the reaction by the heavy metals Cd, Zn, Ni, and Pb at concentrations of 100 mg/l is of concern because these metals are often present in contaminated soils. H₂O₂ has a dominant role in optimizing peroxidase activity in crude horseradish extracts. Fluctuations in temperature and pH, normally experienced in soils, did not appear to have a detrimental impact on peroxidase activity. However, the functioning of the enzyme is seriously affected at a pH ≤ 3. All reactions in this study were carried out in solution.     

Removal of phenols from mixtures by co-immobilized laccase/tyrosinase and Polyclar adsorption

An enzymatic method for removal of phenols from their mixtures was investigated. Phenols in an aqueous solution were removed after a two-step treatment with co-immobilized laccase and tyrosinase and Polyclar (polyvinylpolypyrrolidone). A laccase from Pyricularia oryzae and mushroom tyrosinase were co-immobilized on Mikroperl in a fixed-bed tubular bioreactor by a rapid and simple method. The support immobilized 95% of the total laccase units and 35% of the total tyrosinase units. Different mixtures of phenols were passed through the column with co-immobilized laccase and tyrosinase. This method removed 42–90% of different phenolic substances by a single passage through the bioreactor. The second step employed Polyclar for additional removal of phenolic substances from mixtures. The degree of removal depends on the nature of the phenols. Complete removal was achieved for a-naphthol, 2,4-dichlorophenol, 4-methoxyphenol, b-naphthol, 4-chloro-3-methylphenol and catehin. The operational stability of the immobilized system was 10–90 h depending on the substrate. The biocatalyst was capable of continuous transformation of different phenols in mixtures. Journal of Industrial Microbiology & Biotechnology (2000) 24, 383–388. Received 12 August 1999/ Accepted in revised form 18 February 2000     

Chlorophenol Hydroxylases Encoded by Plasmid pJP4 Differentially Contribute to Chlorophenoxyacetic Acid Degradation

Phenoxyalkanoic compounds are used worldwide as herbicides. Cupriavidus necator JMP134(pJP4) catabolizes 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), using tfd functions carried on plasmid pJP4. TfdA cleaves the ether bonds of these herbicides to produce 2,4-dichlorophenol (2,4-DCP) and 4-chloro-2-methylphenol (MCP), respectively. These intermediates can be degraded by two chlorophenol hydroxylases encoded by the tfdB_I and tfdB_II genes to produce the respective chlorocatechols. We studied the specific contribution of each of the TfdB enzymes to the 2,4-D/MCPA degradation pathway. To accomplish this, the tfdB_I and tfdB_II genes were independently inactivated, and growth on each chlorophenoxyacetate and total chlorophenol hydroxylase activity were measured for the mutant strains. The phenotype of these mutants shows that both TfdB enzymes are used for growth on 2,4-D or MCPA but that TfdB_I contributes to a significantly higher extent than TfdB_II. Both enzymes showed similar specificity profiles, with 2,4-DCP, MCP, and 4-chlorophenol being the best substrates. An accumulation of chlorophenol was found to inhibit chlorophenoxyacetate degradation, and inactivation of the tfdB genes enhanced the toxic effect of 2,4-DCP on C. necator cells. Furthermore, increased chlorophenol production by overexpression of TfdA also had a negative effect on 2,4-D degradation by C. necator JMP134 and by a different host, Burkholderia xenovorans LB400, harboring plasmid pJP4. The results of this work indicate that codification and expression of the two tfdB genes in pJP4 are important to avoid toxic accumulations of chlorophenols during phenoxyacetic acid degradation and that a balance between chlorophenol-producing and chlorophenol-consuming reactions is necessary for growth on these compounds.     

Selection of non-conventional yeasts and their use in immobilized form for the bioremediation of olive oil mill wastewaters

The yeast population dynamics in olive wastewaters (OMW), sampled in five mills from Salento (Apulia, Southern Italy), were investigated. Three hundred yeasts were isolated in five industrial mills and identified by molecular analysis. Strains belonging to Geotrichum, Saccharomyces, Pichia, Rhodotorula and Candida were detected. Five G. candidum strains were able to grow in OMW as the sole carbon source and to reduce phenolics, chemical oxygen demand (COD) and antimicrobial compounds. One G. candidum isolate was selected for whole-cell immobilization in calcium alginate gel. The COD and phenolic reduction obtained with immobilized cells showed a 2.2- and 2-fold increase compared to the removal obtained with free cells, respectively. The immobilization system enhanced yeast oxidative activity by avoiding the presence of microbial protease in treated OMW. To our knowledge, this is the first report on G. candidum whole-cell immobilization for OMW bioremediation.     

Salicylhydroxamic acid enhances the NADH-oxidase activity of peroxidase in pea mitochondrial and chloroplast suspensions

Salicylhydroxamic acid (SHAM), an alternative oxidase inhibitor of plant mitochondria, enhances the NADH-oxidase activity in mitochondrial and chloroplast suspensions obtained from pea roots or leaves, respectively. This reaction is inhibited by the washing of mitochondria or chloroplasts and is observed in supernatants after the removal of the organelles by centrifugation. The reaction is sensitive to CN^– and to antioxidant propyl gallate. The NADH oxidation is also enhanced by 2,4-dichlorophenol or phenol, but not salicylic acid. The acceleration of NADH oxidation by phenolic compounds is observed with presence of commercial horseradish peroxidase and is connected with the involvement of these compounds in NADH-dependent peroxidase reaction. SHAM and 2,4-dichlorophenol significantly enhance the destruction of nuclei in guard cells of pea leaf epidermis caused by the generation of reactive oxygen species during the oxidation of exogenous NADH by apoplastic peroxidase.     

Biodegradation of mixed phenolic compounds by Aspergillus awamori NRRL 3112

The ability of the fungus Aspergillus awamori NRRL 3112 to degrade mixtures of some common phenolic compounds, namely phenol, catechol, 2,4-dichlorphenol and 2,6-dimethoxyphenol was investigated in the present study. For all combinations in which dichlorophenol was incorporated, it took equal time for the nearly complete degradation of the compound—4 days. Phenol was decomposed almost completely (99.5%) in a combination with dimethoxyphenol, to a lesser extent (88%) in a combination with catechol and to the least degree (25%) in the presence of 2,4-dichlorophenol. Catechol experienced a more substantial biotransformation (64%) when mixed with phenol and weaker (45%)—in a combination with dichlorophenol. 2,6-Dimethoxyphenol was better decomposed (69%) in mixtures containing phenol, while its biodegradation in a combination with 2,4-dichlorophenol was considerably poor (only 5%).     

Laccase-mediated detoxification of phenolic compounds

The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined. The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol. A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase. The medium was then inoculated with R. praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis. The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol. Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid. The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions. The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.     

Laccase-mediated detoxification of phenolic compounds.

The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined. The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol. A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase. The medium was then inoculated with R. praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis. The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol. Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid. The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions. The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.     

Bioremediation of phenolic compounds having endocrine-disrupting activity using ozone oxidation and activated sludge treatment

The bioremediation of water system contaminated with phenolic compounds having endocrine-disrupting activity,i.e. 2,4-dichlorophenol, 2,4-dichlorophenoxy acetic acid (2,4-D), and 2,4,5-trichlorophenoxy acetic acid (2,4,5-T), was investigated by using ozone oxidation and activated sludge treatment. Ozone oxidation (ozonation time: 30 min) followed by activated sludge treatment (incubation time: 5 days) was an efficient treatment method for the conversion of phenolic compounds in water into carbon dioxide and decreased the value of total organic carbon (TOC) up to about 10% of initial value. Furthermore, 2,4-D was dissolved in water containing salt,i.e. artificial seawater (ASW), and this water was used as model coastal water contaminated with phenolic compounds. The activated sludge treatment (incubation time: 5 days) could consume significantly organic acids produced from 2,4-D in the model costal water by the ozone oxidation (ozonation time: 30 min) and decrease the value of TOC up to about 35% of initial value.     

Removal of α-naphthol and other phenolic compounds from polluted water by white radish (Raphanus sativus) peroxidase in the presence of an additive, polyethylene glycol

Role of white radish peroxidase has been investigated in the treatment of water contaminated with phenols, particularly α-naphthol. Water polluted with α-naphthol was treated with white radish peroxidase under various experimental conditions. The treatment of α-naphthol polluted water by this enzyme in presence of polyethylene glycol enhanced its removal. Studies carried out in absence of polyethylene glycol showed only 36% of α-naphthol removal however, 96% of it was removed in presence of 0.1 mg/mL of polyethylene glycol in 100 mM sodium phosphate buffer, pH 6.5, and 0.75 mM H₂O₂ at 40°C. The other phenols oxidized and removed from waste water under similar experimental conditions were 18%, m-cresol; 30%, p-chlorophenol; 62%, p-bromophenol; 20%, benzyl alcohol; 21%, quinol; 38%, 2,6-dichlorophenol; 13%, 2,4-dichlorophenol; and 2%, native phenol. Mixtures of different phenolic compounds removed under identical treatment conditions were 63%, A; 40%, B; 52%, C; 41%, D; 72%, E; 66%, F; and 72%, G. Thus, peroxidase in presence of an additive, polyethylene glycol could be a suitable tool for the removal of phenolic compounds from industrial effluents.     

Biodegradation of different molecular-mass polyphenols derived from olive mill wastewaters by Geotrichum candidum

The decolourisation of fresh and stored olive mill wastewaters (OMW) and the biodegradation of three groups (F1, F2 and F3) of phenolic compounds by Geotrichum candidum were investigated. Separated phenolic compounds derived from natural OMW ultrafiltration using membranes with a cutoff 2and 100 kDa. G. candidum growth on fresh OMW decreased pH and reduced COD and colour of 75% and 65%, respectively. However, on the stored-black OMW a failure of COD and colour removal were observed. G. candidum activity on this later substrate was enhanced by the addition of a carbon source easily metabolised, misleading an improvement of the COD reduction and decolourization that reached 58% and 48%, respectively. Growth of G. candidum in the presence of F2 or F3 polyphenolic fractions induced high decolourisation and depolymerisation of phenolic compounds. Whereas, very week decolourisation and biodegradation were observed with F1 fraction. Moreover, the highest levels of lignin peroxidase (LiP) and manganese peroxidase (MnP) activities were obtained in the presence of F2 fraction. These results showed that increasing of molecular-mass of aromatics led to an increase in levels of depolymerisation, decolourisation and COD removal by G. candidum culture.     

Transfer RNA-Peroxidase Interaction: INHIBITION OF INDOLE-3-ACETIC ACID OXIDATION

Transfer RNA from wheat germ, yeast, and Escherichia coli inhibited the indoleacetic acid (IAA)-induced spectral change in horseradish peroxidase (EC 1.11.1.7) and the decarboxylation of IAA. The inhibition was limited to a delay after which the increase in A₄₂₇ and the decarboxylation of IAA resumed at the same rate as in the control; the duration of the inhibition was dependent on, but not proportional to, the concentration of tRNA. Alkaline hydrolysis destroyed the inhibitory activity of tRNA. The inhibition was completely abolished when the tRNA was added 30 seconds after IAA. Thus, the tRNA appears not to react with the enzyme intermediates formed during the reaction with IAA. The inhibition by tRNA was rapidly reversed by H₂O₂ or additional IAA, but not by 2,4-dichlorophenol. Results suggest that the tRNA interferes with the initial reaction between IAA and the heme moiety of free peroxidase, thus preventing the formation of highly active enzyme intermediates essential for IAA degradation.     

Degradation of 4-chloro-2-methylphenol by an activated sludge isolate and its taxonomic description

The Gram-negative strain S1, isolated from activated sludge, metabolized 4-chloro-2-methylphenol by an inducible pathway via a modifiedortho-cleavage route as indicated by a transiently secreted intermediate, identified as 2-methyl-4-carboxymethylenebut-2-en-4-olide by gas chromatography/mass spectrometry. Beside 4-chloro-2-methylphenol only 2,4-dichlorophenol and 4-chlorophenol were totally degraded, without an accumulation of intermediates. The chlorinated phenols tested induced activities of 2,4-dichlorophenol hydroxylase and catechol 1,2-dioxygenase type II. Phenol itself appeared to be degraded more efficiently via a separate, inducibleortho-cleavage pathway. The strain was characterized with respect to its physiological and chemotaxonomic properties. The fatty acid profile, the presence of spermidine as main polyamine, and of ubiquinone Q-10 allowed the allocation of the strain into the -2 subclass of theProteobacteria. Ochrobactrum anthropi was indicated by fatty acid analysis as the most similar organism, however, differences in a number of physiological features (e.g. absence of nitrate reduction) and pattern of soluble proteins distinguished strain S1 from this species.     

Biodegradation of mixed phenolic compounds by Aspergillus awamori NRRL 3112

The ability of the fungus Aspergillus awamori NRRL 3112 to degrade mixtures of some common phenolic compounds, namely phenol, catechol, 2,4-dichlorphenol and 2,6-dimethoxyphenol was investigated in the present study. For all combinations in which dichlorophenol was incorporated, it took equal time for the nearly complete degradation of the compound—4 days. Phenol was decomposed almost completely (99.5%) in a combination with dimethoxyphenol, to a lesser extent (88%) in a combination with catechol and to the least degree (25%) in the presence of 2,4-dichlorophenol. Catechol experienced a more substantial biotransformation (64%) when mixed with phenol and weaker (45%)—in a combination with dichlorophenol. 2,6-Dimethoxyphenol was better decomposed (69%) in mixtures containing phenol, while its biodegradation in a combination with 2,4-dichlorophenol was considerably poor (only 5%).     

Immobilization of Rhizopus oryzae lipase on magnetic Fe3O4-chitosan beads and its potential in phenolic acids ester synthesis

A novel and simple method was developed for the preparation of magnetic Fe₃O₄ nanoparticles by chemical co-precipitation method and subsequent coating with 3-aminopropyltrimethoxysilane (APTMS) through silanization process. Magnetic Fe₃O₄-chitosan particles were prepared by the suspension cross-linking and covalent technique to be used in the application of magnetic carrier technology. The synthesized immobilization supports were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). Using glutaraldehyde as the coupling agent, the lipase from R. oryzae was successfully immobilized onto the functionalized magnetic Fe₃O₄-chitosan beads. The results showed that 86.60% of R. oryzae lipase was bound on the synthesized immobilization support. This immobilized lipase was successfully used for the esterification of phenolic acid which resulted in esterification of phenolic acid in isooctane solvent reaction system for 8 consecutive cycles (totally 384 h), 72.6% of its initial activity was retained, indicating a high stability in pharmaceutical and industrial applications.     

Interactions between rhamnolipid biosurfactants and toxic chlorinated phenols enhance biodegradation of a model hydrocarbon-rich effluent

Surfactant-mediated treatment increases hydrocarbon solubilization and potentially facilitates biodegradation, unless toxic co-contaminants inhibiting microbial activity are present in the hydrocarbon mixture. We assessed the effect of rhamnolipids on the performance of a bacterial consortium degrading diesel fuel employed as a model hydrocarbon-rich effluent, co-contaminated with toxic phenol, 4-chlorophenol (4-CP) or 2,4-dichlorophenol (2,4-DCP). This approach led to the unexpected finding that rhamnolipids reduced toxicity of 4-CP and 2,4-DCP to the hydrocarbon-degrading cells. The facts that rhamnolipids decreased diesel fuel - water partition coefficient (K_FW) of 4-CP and 2,4-DCP and modified aggregate size distribution profiles of the dispersed diesel fuel - chlorinated phenols solutions, suggest the existence of specific interactions between rhamnolipids and the co-contaminants. Due to the polar nature of 4-CP and 2,4-DCP, possible explanations involve adsorption of 4-CP and 2,4-DCP on the surface of biosurfactant aggregates. This property of rhamnolipids is of interest to those using biosurfactants for microbial treatment of hydrocarbon-rich wastewaters co-contaminated with toxic compounds.     

Oxidase reactions of tomato anionic peroxidase

Tomato (Lycopersicon esculentum Mill) anionic peroxidase was found to catalyze oxidase reactions with NADH, glutathione, dithiothreitol, oxaloacetate, and hydroquinone as substrates with a mean activity 30% that of horseradish peroxidase; this is in contrast to the negligible activity of the tomato enzyme as compared to the horseradish enzyme in catalyzing an indoleacetic acid-oxidase reaction with only Mn²⁺ and a phenol as cofactors. Substitution of Ce³⁺ for Mn²⁺ produced an 18-fold larger response with the tomato enzyme than with the horseradish enzyme, suggesting a significant difference in the autocatalytic indoleacetic acid-oxidase reactions with these two enzymes. In attempting to compare enzyme activities with 2,4-dichlorophenol as a cofactor, it was found that reaction rates increased exponentially with both increasing cofactor concentration and increasing enzyme concentration. While the former response may be analogous to allosteric control of enzyme activity, the latter response is contrary to the principle that reaction rate is proportional to enzyme concentration, and additionally makes any comparison of enzyme activity difficult.