Benzoate degradation by Rhodococcus opacus 1CP after dormancy: Characterization of dioxygenases involved in the process

The process of benzoate degradation by strain Rhodococcus opacus 1CP after a five-year dormancy was investigated and its peculiarities were revealed. The strain was shown to be capable of growth on benzoate at a concentration of up to 10 g L^?1. The substrate specificity of benzoate dioxygenase (BDO) during the culture growth on a medium with a low (200–250 mg L^?1) and high (4 g L^?1) concentration of benzoate was assessed. BDO of R. opacus 1CP was shown to be an extremely narrow specificity enzyme. Out of 31 substituted benzoates, only with one, 3-chlorobenzoate, its activity was higher than 9% of that of benzoate. Two dioxygenases, catechol 1,2-dioxygenase (Cat 1,2-DO) and protocatechuate 3,4-dioxygenase (PCA 3,4-DO), were identified in a cell-free extract, purified and characterized. The substrate specificity of Cat 1,2-DO isolated from cells of strain 1CP after the dormancy was found to differ significantly from that of Cat 1,2-DO isolated earlier from cells of this strain grown on benzoate. By its substrate specificity, the described Cat 1,2-DO was close to the Cat 1,2-DO from strain 1CP grown on 4-methylbenzoate. Neither activity nor inhibition by protocatechuate was observed during the reaction of Cat 1,2-DO with catechol, and catechol had no inhibitory effect on the reaction of PCA 3,4-DO with protocatechuate.     

Aerobic degradation of di- and trichlorobenzenes by two bacteria isolated from polluted tropical soils

Two polychlorinated biphenyl (PCBs)-degrading bacteria were isolated by traditional enrichment technique from electrical transformer fluid (Askarel)-contaminated soils in Lagos, Nigeria. They were classified and identified as Enterobacter sp. SA-2 and Pseudomonas sp. SA-6 on the basis of 16S rRNA gene analysis, in addition to standard cultural and biochemical techniques. The strains were able to grow extensively on dichloro- and trichlorobenzenes. Although they failed to grow on tetrachlorobenzenes, monochloro- and dichlorobenzoic acids, they were able to utilize all monochlorobiphenyls, and some dichlorobiphenyls as sole sources of carbon and energy. The effect of incubation with axenic cultures on the degradation of 0.9 mM 1,4-dichlorobenzene, 0.44 mM 1,2,3- and 0.43 mM 1,3,5-trichlorobenzene in mineral salts medium was studied. Approximately, 80-90% of these xenobiotics were degraded in 200 h, concomitant with cell increase of up to three orders of magnitude, while generation times ranged significantly (P<0.05) from 17-32 h. Catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities were detected in crude cell-free extracts of cultures pre-grown with benzoate, with the latter enzyme exhibiting a slightly higher activity (0.15-0.17 micromolmin(-1) mg of protein(-1)) with catechol, suggesting that the meta-cleavage pathway is the most readily available catabolic route in the SA strains. The wider substrate specificity of these tropical isolates may help in assessing natural detoxification processes and in designing bioremediation and bioaugmentation methods.     

Detection of dioxygenase genes present in various activated sludge

GOAL, SCOPE AND BACKGROUND: Activated sludge from refineries contains various microorganisms that could utilize aromatics under aerobic conditions due to the oxygenase enzymes. Dioxygenase enzymes are oxygenases, which are involved in the ring cleavage step of aromatic hydrocarbons. In this study, the selected catabolic loci involved in ring cleavage have been monitored in the activated sludge samples at different time intervals. The investigation of the dioxygenase genes in the Effluent Treatment Plants (ETPs) and evaluation of their presence at different time points provides a clue for the aromatic utilizing potential of the inherent microbial flora. METHODS: The catabolic gene loci pheB, xylE, tod-isp, bed and nahG responsible for the enzymes catechol 1,2-dioxygenase, catechol 2,3-dioxygenase, toluene dioxygenase-iron-sulphur protein component, benzene dioxygenase and naphthalene dioxygenase were used respectively. The time dependent change in eubacterial population was demonstrated by the amplification of 16S rDNA product, followed by restriction digestion. The template DNA was obtained from the activated sludge collected from ETPs. The supporting physiological data for the overall performance of sludge was developed using respirometric analysis. The on-site COD and MLSS analysis for ETP was used in final evaluation. The study was carried out with samples collected from three different ETPs and also from a selected ETP at different time intervals. RESULTS AND DISCUSSION: The respirometric studies were carried out with phenol, catechol, toluene, and naphthalene to arrive at the target genotypes for further study by PCR protocol. The respirometric analysis coupled with the COD and MLSS analysis represented the physiological capacity of the various sludges. Initially, the tracking protocol was optimized by using different sludge samples, which were collected from refineries. The selected genotypes were amplified and their presence has been confirmed using Southern analysis. The gene loci tod-isp, bed and xylE were commonly observed at various time intervals of the sludge from the same source. The gene loci pheB and nahG were found to be relatively rare. CONCLUSION: The 16S rDNA PCR products after restriction digestion produced different DNA fingerprint patterns, suggesting that the microbial community composition was diverse in the three sources. Similarly, the presence of the catechol 2,3-dioxygenase, benzene dioxygenase and toluene dioxygenase genes confirmed the aromatic degrading potential in the various sludges. The probes could not pick the nahG and pheB genes. However, the respirometeric assay suggested that the oxidative capacity to use naphthalene as a substrate exists. RECOMMENDATION AND PERSPECTIVE: Our study of the diversity at various time points from the ETP provided an overview of the shifts of the catabolic composition of the sludge. This also depends on the influential parameters like the incoming pollutant level and the environmental conditions that are prevailing and often changing from time to time. The results of direct DNA extraction and PCR amplification do reflect the relative abundance of a particular catabolic genotype, which could be used to monitor the efficiency of treatment.     

Degradation of 3,4-dichloro- and 3,4-difluoroaniline by Pseudomonas fluorescens 26-K

3,4-Dichloro- and 3,4-difluoroanilines were degraded by Pseudomonas fluorescens 26-K under aerobic conditions. In the presence of glucose strain degraded 170 mg/L of 3,4-dichloroaniline (3,4-DCA) during 2-3 days. Increasing of toxicant concentration up to 250 mg/L led to degradation of 3,4-DCA during 4 days and its intermediates during 5-7 days. Without cosubstrate and nitrogen source degradation of 3,4-DCA took place too, but more slowly--about 40% of toxicant at initial concentration 75 mg/L was degraded during 15 days. 3,4-Difluoroaniline (3,4-DFA) (initial concentration 170 mg/L) was degraded by Pseudomonas fluorescens 26-K during 5-7 days. The strain was able to completely degrade up to 90 mg/L of 3,4-DFA, without addition of cosubstrate and nitrogen during 15 days. Degradation of fluorinated aniline was accompanied by intensive defluorination. Activity of catechol 2,3-dioxygenase (C2,3DO) (0.230 micromol/min/mg of protein) was found in the culture liquid of the strain, grown with 3,4-DCA and glucose. This fact, as well as, the presence of 3-chloro-4-hydroxyaniline as a metabolite suggested that 3,4-DCA degradation pathway includes dehalogenation and hydroxylation of aromatic ring followed by its subsequent cleaving by C2,3DO. On the contrary, activity of catechol 1,2-dioxygenase (C1,2DO) (0.08 micromol/min/mg of protein) was found in the cell-free extract of biomass grown on 3,4-DFA. 3-Fluoro-4-hydroxyaniline as intermediate was found in this cell-free extract.     

Mutant AFM 2 of Alcaligenes faecalis for phenol biodegradation using He-Ne laser irradiation

He-Ne laser technology was utilized in this study to investigate the response of Alcaligenes faecalis to laser stimulation. The irradiation experiments were conducted by the adjustment of the output power from 5 to 25 mW and the exposure time from 5 to 25 min. The results showed that the survival rate changed regularly with the variety of irradiation dose, and high positive mutation frequency was determined by both the energy density and the output power. The mutant strain AFM 2 was obtained. Phenol biodegradation assay demonstrated that AFM 2 possessed a more prominent phenol-degrading potential than its parent strain, which presumably attributed to the improvements of phenol hydroxylase and catechol 1,2-dioxygenase activities. The phenol of 2000 mgl(-1) was completely degraded by AFM 2 within 85.5h at 30 degrees C. In addition, the cell growth and phenol degradation kinetics of the mutant strain AFM 2 and its parent strain in batch cultures were also investigated at the wide initial phenol concentration ranging from 0 to 2000 mgl(-1) by Haldane model. The results of these experiments further demonstrated that the mutant strain AFM 2 possessed a higher capacity to resist phenol.     

Bioremediation of environmental endocrine disruptor di-n-butyl phthalate ester by Rhodococcus ruber

In this study DBP-degradation strain CQ0301 was isolated from rubbish landfill soil. According to the biophysical, biochemical characteristics and analysis of 16S rRNA, the strain was identified as Rhodococcus ruber. Three new protein bands could be fractioned after DBP-inducing, which were suspected to participate the process of DBP-degrading. Catechol was suspected to be an intermediate product of DBP and cleaving the benzene ring was catalyzed by catechol 1,2-dioxygenase, because a highly activity of catechol 1,2-dioxygenase could be detected after DBP-inducing. The results of this study also showed the optimal pH value, optimal temperature which influenced the degradation rate in soil: pH 7.0-8.0, 30-35 degrees C. Kinetics of degradation reaction had been performed at different initial concentration and different time. Analyzed with SPSS10.0 software, the DBP degradation can be described as the same exponential model when the initial DBP concentration was lower than 50 mg/kg. The kinetics equation was lnC=-0.1332t + A, with the degradation half-life of DBP in soil (5.20 d). Inoculating CQ0301 could relieve DBP content in plant. We also found that adding nutrient materials into soil was useful for decreasing the DBP content in plant. In summary, we isolated a bacterium capable of degrading DBP and decreasing DBP content in plant. We also explored the mechanism of biodegradation and characterized the environmental factors influencing the degradation process in contaminated soil. Based on this work, we hope that these findings can provide some information for applying of bioremediation of DBP contaminated soil.     

Naphthalene metabolism in Nocardia otitidiscaviarum strain TSH1, a moderately thermophilic microorganism

The thermophilic bacterium Nocardia otitidiscaviarum strain TSH1, originally isolated in our laboratory from a petroindustrial wastewater contaminated soil in Iran, grows at 50 degrees C on a broad range of hydrocarbons. Transformation of naphthalene by strain TSH1 which is able to use this two ring-polycyclic aromatic hydrocarbon (PAH) as a sole source of carbon and energy was investigated. The metabolic pathway was elucidated by identifying metabolites, biotransformation studies and monitoring enzyme activities in cell-free extracts. The identification of metabolites suggests that strain TSH1 initiates its attack on naphthalene by dioxygenation at its C-1 and C-2 positions to give 1,2-dihydro-1,2-dihydroxynaphthalene. The intermediate 2-hydroxycinnamic acid, characteristic of the meta-cleavage of the resulting diol was identified in the acidic extract. Apart from typical metabolites of naphthalene degradation known from mesophiles, benzoic acid was identified as an intermediate for the naphthalene pathway of this Nocardia strain. Neither phthalic acid nor salicylic acid metabolites were detected in culture extracts. Enzymatic experiments with cell extract showed the catechol 1,2-dioxygenase activity while transformation of phthalic acid and protocatechuic acid was not observed. The results of enzyme activity assays and identification of benzoic acid in culture extract provide strong indications that further degradation goes through benzoate and beta-ketoadipate pathway. Our results indicate that naphthalene degradation by thermophilic N. otitidiscaviarum strain TSH1 differs from the known pathways found for the thermophilic Bacillus thermoleovorans Hamburg 2 and mesophilic bacteria.     

Stereoselective biosynthesis of chloroarylpropane diols by the basidiomycete Bjerkandera adusta

Previously we have shown that 1-arylpropane-1,2-diols are catabolic products of L-phenylalanine during idiophasic metabolism of B. adusta that are stereoselectively biosynthesized from a C(7)-unit (ring+benzylic carbon) and a C(2)-unit as predominantly erythro 1R, 2S enantiomers.In order to probe the mechanism of 1-arylpropane-1,2-diol formation, the products of the incubation of isotopically labelled aromatic aldehydes as substrates with Bjerkandera adusta (DAOM 215869) have been characterized. The aromatic aldehydes were benzaldehyde (ring D(5)) and 4-methoxy- and 4-hydroxybenzaldehydes (ring 13C(6)). These aldehydes were all stereoselectively incorporated into the corresponding 1-arylpropane-1,2-diols, including the chloro analogues, as well as into the corresponding alpha-ketols (phenyl acetyl carbinols (PAC's) and 2-hydroxy propiophenones (2-HPP's)) the presumed precursors of the diols. Benzoic acid (ring D(5)) was likewise incorporated into the diols, chlorodiols and alpha-ketols.These results lead us to conclude that the aromatic aldehydes benzaldehyde, 4-hydroxybenzaldehyde and 4-methoxybenzaldehyde are likely C(7)-unit precursors in the carboligation reaction(s) that leads to 1-arylpropane-1,2-diol biosynthesis. The metabolic role of the diols remains to be elucidated but they may be important intermediates in CAM (chlorinated anisyl metabolite) aldehyde-alcohol cycling and also act as substrates for the chlorination/hydroxylation enzymes yet to be identified in white rot fungi.     

Reaction of spruce cells toward heavy metals and the influence of culture conditions

BACKGROUND: Plant cell cultures may serve as biosensors for the detection of heavy metals and other toxic substances. Standard culture media and protocols are frequently utilised, but in these media no care is usually taken to control the influence of hormones and nutrients on the reaction of the enzymes or m under consideration as parts of the sensor. The present paper investigates the influence of media composition on the reaction of spruce cells towards heavy metals. METHODS: Spruce cell cultures were grown in a standard medium, either i) alone, ii) containing 0.3% sucrose or iii) containing 3% sucrose and the hormones BAP and NAA. The cell cultures were then incubated in medium with fungal elicitor, H2O2, CdSO4 (50 to 500 microM), or, alternatively, with a standard heavy metal mixture containing 80 microM Na2HAsO4, 150 microM CdSO4 and 200 microM PbCl2. RESULTS: Depending on the nutrient status and hormone availability, large differences in glutathione contents and the GSH/GSSG ratio were observed. However, the cellular redox state seemed to remain more or less constant. Glutathione S-transferase activity was determined with four substrates, and high induction rates for the conjugation of three substrates were observed when hormones were omitted from the media. 1,2-epoxy-nitrophenoxy-propane conjugation was highest in starving cell in the presence of hormones, showing a transient GST induction, with highest rates occurring after 16 hrs following incubation; the induction effect was lost after 24 hrs. CONCLUSION: A medium containing 3% sucrose and both hormones (BAP and NAA) appears to be most favourable for cellular growth as well as the expression of a basis level of detoxification enzymes and antioxidants. With this combination, early responses towards heavy metals at low concentration can be monitored. RECOMMENDATIONS AND PERSPECTIVE: Plant cell cultures are valuable tools for the bioindication of heavy metals and toxic xenobiotics. If standard media and protocols are utilised, the influence of hormones and nutrients on the reaction of the biosensor have to be evaluated thoroughly.     

Mechanistic insights into the specificity of human cytosolic sulfotransferase 2A1 (hSULT2A1) for hydroxylated polychlorinated biphenyls through the use of fluoro-tagged probes

Determining the relationships between the structures of substrates and inhibitors and their interactions with drug-metabolizing enzymes is of prime importance in predicting the toxic potential of new and legacy xenobiotics. Traditionally, quantitative structure activity relationship (QSAR) studies are performed with many distinct compounds. Based on the chemical properties of the tested compounds, complex relationships can be established so that models can be developed to predict toxicity of novel compounds. In this study, the use of fluorinated analogues as supplemental QSAR compounds was investigated. Substituting fluorine induces changes in electronic and steric properties of the substrate without substantially changing the chemical backbone of the substrate. In vitro assays were performed using purified human cytosolic sulfotransferase hSULT2A1 as a model enzyme. A mono-hydroxylated polychlorinated biphenyl (4-OH PCB 14) and its four possible mono-fluoro analogues were used as test compounds. Remarkable similarities were found between this approach and previously published QSAR studies for hSULT2A1. Both studies implicate the importance of dipole moment and dihedral angle as being important to PCB structure in respect to being substrates for hSULT2A1. We conclude that mono-fluorinated analogues of a target substrate can be a useful tool to study the structure activity relationships for enzyme specificity.     

Effects of some insecticides on several enzymes of tryptophan metabolism in rats

The activities of tryptophan 2,3-dioxygenase (EC 1.13.11.11), indoleamine 2,3-dioxygenase (EC 1.13.11.17), kynurenine 3-hydroxylase (EC 1.14.13.9), kynureninase (EC 3.7.1.3), kynurenine transaminases, and pyridoxal phosphokinase (EC 2.7.1.35) in the liver, kidney and lung rats were measured after administration of a single dose and repeated doses of dimethoate, carbaryl and fenvalerate, respectively. Ten percent LD50 of each insecticide was orally administered to a rat for a single dose, while 5% LD50 was orally given for five consecutive days as repeated doses. The control animals received the same volume of vehicle (polyethylene glycol 300). Body weight and organs weight losses were recognized only after repeated doses of dimethoate, while protein content remained constant compared to control animals. Repeated administration of dimethoate caused significant decrease in the activity of kynurenine 3-hydroxylase (28.3% decrease in liver, and 32.5% in kidney), kynurenine-pyruvate transaminase (EC 2.6.1.7) (40% in liver, and 24.2% in kidney), kynurenine- pyruvate transaminase (EC 2.6.1-) (24.5% in kidney) and pyridoxal phosphokinase (36.1% in liver). Repeated doses of carbaryl resulted in a significant decrease in the activity of apo-tryptophan 2,3-dioxygenase (42.8%), kynurenine-2-oxoglutarate transaminase (40% in liver), kynurenine-pyruvate transaminase (30.6% in liver), and serine-glyoxylate transaminase (EC 2.6.1.51) (47.9% in liver). Externally added insecticides at different concentrations to the incubation mixture resulted in an inhibition to tryptophan 2,3-dioxygenase, while the other enzymes examined showed no change in their activities.     

Aerosol formation yields from the reaction of catechol with ozone

The formation of secondary organic aerosol from the gas-phase reaction of catechol (1,2-dihydroxybenzene) with ozone has been studied in two smog chambers. Aerosol production was monitored using a scanning mobility particle sizer and loss of the precursor was determined by gas chromatography and infrared spectroscopy, whilst ozone concentrations were measured using a UV photometric analyzer. The overall organic aerosol yield (Y) was determined as the ratio of the suspended aerosol mass corrected for wall losses (M_o) to the total reacted catechol concentrations, assuming a particle density of 1.4 g cm^?3. Analysis of the data clearly shows that Y is a strong function of M_o and that secondary organic aerosol formation can be expressed by a one-product gas–particle partitioning absorption model. The aerosol formation is affected by the initial catechol concentration, which leads to aerosol yields ranging from 17% to 86%. The results of this work are compared to similar studies reported in the literature.     

Novel degradation pathway of 4-chloro-2-aminophenol via 4-chlorocatechol in Burkholderia sp. RKJ 800

Burkholderia sp. RKJ 800 utilized 4-chloro-2-aminophenol (4C2AP) as the sole carbon and energy source and degraded it with release of chloride and ammonium ions. The metabolic pathway of degradation of 4C2AP was studied and a novel intermediate, 4-chlorocatechol was identified as a major degradation product of 4C2AP using high-performance liquid chromatography and gas chromatography–mass spectrometry. Enzyme activities for 4C2AP-deaminase and 4-chlorocatechol-1,2-dioxygenase were detected in the crude extracts of the 4C2AP-induced cells of strain RKJ 800. The activity of the 4C2AP-deaminase confirmed the formation of 4-chlorocatechol from 4C2AP and the 4-chlorocatechol-1,2-dioxygenase activity suggested the cleavage of 4-chlorocatechol into 3-chloro-cis,cis-muconate. On the basis of the identified metabolites, we have proposed a novel degradation pathway of 4C2AP for Burkholderia sp. RKJ 800. Furthermore, the potential of Burkholderia sp. RKJ 800 to degrade 4C2AP in soil was also investigated using microcosm studies under laboratory conditions. The results of microcosm studies conclude that Burkholderia sp. RKJ 800 was able to degrade 4C2AP in soil and may be used to remediate 4C2AP-contaminated site. This is the first report of (1) the formation of 4-chlorocatechol and 3-chloro-cis,cis-muconate in the degradation pathway of 4C2AP and (2) bioremediation of 4C2AP by any bacterium.     

The effect of cyclic aerobic-anoxic conditions on biodegradation of benzoate.

The response of a mixed microbial culture to cyclic aerobic and anoxic (denitrifying) conditions was studied in a chemostat with a 48-hour hydraulic residence time receiving a feed containing benzoate and pyruvate. When the cyclic conditions were 3-hour aerobic and 9-hour anoxic, the bacteria-degraded benzoate aerobically via the catechol 2,3-dioxygenase (C23DO) pathway. The quantity of C23DO remained constant throughout the anoxic period but decreased during the initial portion of the aerobic period before returning to the level present in the anoxic period. Anoxic biodegradation of benzoate was via benzoyl-CoA reductase, which remained constant regardless of the redox condition. The aerobic benzoate uptake capability (AeBUC) of the culture increased during the aerobic period but decreased during the anoxic period. The anoxic benzoate uptake capability (AnBUC) exhibited the opposite response. When the cycle was 6-hour aerobic and 6-hour anoxic, aerobic biodegradation of benzoate proceeded via the protocatechuate 4,5-dioxygenase (P45DO) pathway. The P45DO activity decreased early in the aerobic period, but then increased to the level present during the anoxic period. The level of benzoyl-CoA reductase was constant throughout the cycle. Furthermore, AeBUC and AnBUC responded in much the same way as in the 3/9-hour chemostat. During a 9-hour aerobic and 3-hour anoxic cycle, the culture synthesized both P45DO and C23DO, with the former having significantly higher activity. Unlike the other two cycles, AeBUC changed little during the aerobic period, although AnBUC decreased. The culture was well-adapted to the cyclic conditions as evidenced by the lack of accumulation of either substrate during any cycle tested. This suggests that cyclic aerobic-anoxic processes can be used in industrial wastewater-treatment facilities receiving significant quantities of simple aromatic compounds like benzoate. However, the results showed that the kinetics of benzoate degradation were different under aerobic and anoxic conditions, a situation that must be considered when modeling cyclic bioreactors receiving aromatic compounds.     

Effect of herbicides on glutathione S-transferases in the earthworm, <Emphasis Type="Italic">Eisenia fetida</Emphasis>

AIM AND BACKGROUND: Earthworms have been studied as a readily available, easily maintainable and cheap test species for assessing chemical pollution, and may be an alternative to in vivo rodent bioassays. The current investigation aims to characterize detoxification enzymes in Eisenia fetida and stress response against two herbicides with different modes of action, namely, fenoxaprop and metolachlor. METHODS: Herbicides were applied to soil containing earthworms. Animals were then collected, sacrificed and shock-frozen. Extracted protein was analyzed for glutathione S-transferase (GST) activity using CDNB (1-chloro-2,4-dinitrobenzene), DCNB (1,2-dichloro-4-nitrobenzene), pNBC (p-nitrobenzylchloride), PNOBC (p-nitrobenz-o-ylchloride) and selected herbicides. GST isoenzymes were partially purified by affinity chromatography and molecular weights were estimated by SDS-PAGE. RESULTS: In E. fetida protein extracts, GST activity towards model compounds ranked as CDNB>DCNB>PNBOC>PNBC. Fluorodifen was not conjugated at all, but fenoxaprop and metolachlor were conjugated at low rates. Furthermore, the GST isoenzyme pattern changed during the incubation with herbicides, either due to stress or as a defense reaction. After incubation with monochlorobimane, a strong fluorescence of the intestinal tract and the intersegments was observed, indicating organ-specific GST induction. DISCUSSION: According to the author's knowledge, here, for the first time, evidence is presented that E. fetida GST are also capable of conjugating a wider range of xenobiotic substrates. Different forms of GST were observed and changes in GST isoforms due to the herbicide treatment were also noticed. GST conjugation rates varied between different herbicides used in this experiment. It might be assumed that herbicides may well be detoxified by earthworms, to a certain extent, but that they are also potent stress factors influencing the detoxification system of the animal. High doses or long exposure might lead to deleterious effects on earthworms and limit their survival rate. The use of the animals as bioindicators for herbicides and herbicide residues seems very promising, but is surely influenced by the lack of detoxification for some compounds. CONCLUSIONS: Conjugation of several xenobiotics with model substances and herbicides is proven in the earthworm E. fetida. However, E. fetida has only limited capabilities of detoxifying herbicidal compounds. Different isoforms of GST were involved and altered in their activity after treatment. RECOMMENDATIONS AND PERSPECTIVES: The accumulation of GS-conjugates and their determination via fluorescence microscopy is a quick and secure, additional marker for exposure that should be further developed to complement existing biotests. The described methods and endpoints might help to understand the complex reaction of earthworms towards herbicides and lead to an adapted test methodology.     

Electrochemical oxidation of benzene on boron-doped diamond electrodes

This work presents an electrochemical investigation of the benzene oxidation process in aqueous solution on boron-doped diamond (BDD) electrodes. Additionally, in order to determine the main products generated during the oxidation process, electrolysis and high performance liquid chromatography experiments were carried out. The complete degradation of this compound was performed aiming to a further application in waste water treatment. The cyclic voltammetry studies indicate that benzene is irreversibly oxidized in acid medium (H₂SO₄ 0.5 M) on the BDD electrode surface at 2.0 V versus Ag/AgCl in a diffusion controlled process. During the cycling, other products are generated, and a pair of peaks was observed that can be associated with the oxi-reduction of anyone of the following species: hydroquinone, benzoquinone, resorcinol or catechol. The electrolysis experiments were carried out at 2.4 and 2.5 V on the BDD electrode surface in a solution containing 1 × 10⁻² M of benzene (below the saturation concentration in aqueous solution), for 3 and 5 h, respectively. The main products measured were: hydroquinone, resorcinol, p-benzoquinone, catechol and phenol. The complete electrochemical benzene degradation was performed in the electrolysis experiments using a rotating BDD disc electrode (2.5 V for 5 h) and the main products detected were all measured at concentrations lower than 10⁻⁵ M in this condition. The boron-doped diamond electrode had proved to be a valuable tool for the electrochemical degradation of the benzene, a very stable chemical compound.     

Estimation of biodegradation parameters of phenolic compounds on activated sludge by respirometry

The bio-oxidation of phenol, catechol, resorcinol, m-cresol and 5-methylresorcinol on activated sludge was investigated by oxygen uptake measurements. In addition, the degradation of acetate with the same microbial population was studied. The substrate-dependent oxygen uptake data were analysed on the basis of the Michaelis-Menten kinetics. The extant kinetic parameters, the maximum rates of oxygen consumption and half-saturation constants for the processes with different substrates were determined. The simple respirometric approach also made it possible to determine the short-term oxygen demands of the substrates which formed 23-38% of the theoretical oxygen demand of the studied compounds.     

Biodegradation of catechol (2-hydroxy phenol) bearing wastewater in an UASB reactor

The present study deals with the biodegradation of catechol through co-metabolism with glucose in aqueous solution as primary substrate in an upflow anaerobic sludge blanket (UASB) reactor. Batch studies indicated that the 1000mgl(-1) glucose concentration was sufficient to cometabolize and degrade catechol in an aqueous solution up to a concentration of 1000mgl(-1). The reactor operated at 35+/-2 degrees C, and at a constant hydraulic retention time of 8h with a gradual stepwise increase in catechol concentration from 100 to 1000mgl(-1) along with glucose as a cosubstrate. The results showed that the catechol was successfully mineralized in an UASB reactor in which microbial granulation was achieved with only glucose as the substrate. The reactor showed > or = 95% COD removal efficiency with 500-1000mgl(-1)catechol concentration in the feed and a glucose concentration of 1500mgl(-1) as a cosubstrate. Similar efficiency was obtained at a constant catechol concentration of 1000mgl(-1) with 500-1000mgl(-1) glucose concentration. Once the reactor got acclimatized with catechol, higher concentrations of catechol can be mineralized with a minimum amount of glucose as the cosubstrate without affecting the performance of the UASB reactor.     

Transformation of phenol, catechol, guaiacol and syringol exposed to sodium hypochlorite

Germs, xenobiotics and organic matter that influence the colour, turbidity and organoloeptic properties of water are removed by chlorination. Unfortunately, chlorine oxidants including sodium hypochlorite, used in water treatment induce processes that partly convert the treated compounds to unwanted chlorinated derivatives. The purpose of this work was to analyse the efficiency of transformation of phenol, catechol, guaiacol and syringol exposed to sodium hypochlorite and determine the intermediates formed during oxidative conversion of these compounds. The analysis was performed in aerobic conditions, both in acidic (pH 4.0) and alkaline (pH 8.0) medium. The effectiveness of transformation was slightly higher in acidic in comparison to alkaline conditions. Some chlorophenols, such as 2-chlorophenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol and pentachlorophenol were determined as the products of phenol conversion. Chlorophenols were also formed during catechol, guaiacol and syringol transformation by replacement of hydroxy and methoxy residues by chlorine atoms. Moreover, some chlorocatechols and chlorinated methoxyphenols were determined during catechol and methoxyphenols transformations. Higher concentrations of chlorinated compounds were observed in the alkaline environment during phenol transformation. Conversion of catechol and methoxyphenols generated higher amounts of chlorinated intermediates in the acidic medium. In samples carboxylic acids like acetic and formic acids were determined. The formation of these compounds was the result of the cleavage of aromatic structure of phenols.