Freeze-dried recombinant bacteria for on-site detection of phenolic compounds by color change

We herein report the development of a recombinant bacterial biosensor for the rapid and easy detection of phenolic compounds in the field. A plasmid was designed to encode a beta-galactosidase reporter gene under the control of capR, an activator involved in phenolic compound degradation. The construct was transformed into Escherichia coli, and transformed cells were stored after being freeze-dried in the presence of sucrose. For detection of phenolic compounds, the cells were rehydrated, and used instantly, without any growth step. In the presence of 0.1 microM-10mM phenol, we observed a red color from hydrolysis of chlorophenol red beta-D-galactopyranoside (CPRG) or an indigo color from hydrolysis of X-galactopyranoside (X-gal). Other phenolic compounds could be detected by this system, including catechol, 2-methylphenol, 2-chlorophenol, 3-methylphenol, 2-nitrophenol, and 4-chlorophenol. These results suggest that this novel bacteria biosensor may be useful for easy, on-site detection of phenolic compounds without the need for unwieldy equipment or sample pretreatment. Indeed, biosensor systems involving beta-galactosidase-expressing freeze-dried recombinant bacteria could prove useful for the in situ detection of many more compounds in the future.     

Development of a high analytical performance-tyrosinase biosensor based on a composite graphite-Teflon electrode modified with gold nanoparticles

The design of a new tyrosinase biosensor with improved stability and sensitivity is reported. The biosensor design is based on the construction of a graphite-Teflon composite electrode matrix in which the enzyme and colloidal gold nanoparticles are incorporated by simple physical inclusion. Experimental variables such as the colloidal gold loading into the composite matrix, the enzyme loading and the potential applied to the bioelectrode were optimized. The Tyr-Au(coll)-graphite-Teflon biosensor exhibited suitable amperometric responses at -0.10 V for the different phenolic compounds tested (catechol; phenol; 3,4-dimethylphenol; 4-chloro-3-methylphenol; 4-chlorophenol; 4-chloro-2-methylphenol; 3-methylphenol and 4-methylphenol). The limits of detection obtained were 3 nM for catechol, 3.3 microM for 4-chloro-2-methylphenol, and approximately 20 nM for the rest of phenolic compounds. The presence of colloidal gold into the composite matrix gives rise to enhanced kinetics of both the enzyme reaction and the electrochemical reduction of the corresponding o-quinones at the electrode surface, thus allowing the achievement of a high sensitivity. The biosensor exhibited an excellent renewability by simple polishing, with a lifetime of at least 39 days without apparent loss of the immobilized enzyme activity. The usefulness of the biosensor for the analysis of real samples was evaluated by performing the estimation of the content of phenolic compounds in water samples of different characteristics.     

A new variant activator involved in the degradation of phenolic compounds from a strain of Pseudomonas putida

A new variant type of regulatory activator and relevant promoters (designated capR, Pr and Po) involved in the metabolism of phenolic compounds were cloned from Pseudomonas putida KCTC1452 by using PCR. The deduced amino acid sequence of CapR revealed a difference in nine amino acids from the effector binding domain of DmpR. To measure effector specificity, plasmids were constructed in such a way that the expression of luc gene for firefly luciferase or lacZ for beta-galactosidase as a reporter was under the control of capR. When Escherichia coli transformed with the plasmids was exposed to phenol, dramatic increases in the activity of luciferase or beta-galactosidase were observed in a range of 0.01-1 mM. Among various phenolic compounds tested, other effective compounds included catechol, 2-methylphenol, 3-methylphenol, 4-methylphenol, 2-chlorophenol, 4-chlorophenol, 2-nitrophenol, resorcinol, and 2, 5-dimethylphenol. The results indicate that CapR has effector specificity different from other related activators, CatR and DmpR. Waste water and soil potentially containing phenolic compounds were also tested by this system and the results were compared with chemical and GC data. The present results indicate that the biosensor consisting of capR and the promoters may be utilized for the development of a phenolic compounds-specific biosensor in monitoring the environmental pollutant.     

Influence of phenols on growth and membrane permeability of free and immobilized Escherichia coli.

The inhibition of the exponential growth of Escherichia coli K-12 by different phenolic compounds was examined. Cells entrapped in calcium alginate showed a greater tolerance than cells grown in suspension. The extent of inhibition of growth of the immobilized cells depended on the period of growth in the gel matrix. After the addition of bacteriostatic concentrations of phenol or 4-chlorophenol, a dose-dependent efflux of metabolites such as ATP and of K+ ions was elicited. Provided that glucose was supplied as an energy substrate, a reaccumulation of K+ ions at low phenol concentrations was observed. The restoration of the membrane gradient for K+ always preceded the continuation of growth in the presence of the toxic compounds. Compared with free cells, those cells immobilized and grown in alginate suffered a smaller loss of cations after the addition of 4-chlorophenol. The reestablishment of gradients was observed at higher concentrations of the pollutants with entrapped cells than with free cells. Corresponding to the increase in tolerance, the membrane damage was reduced in cells grown in immobilized form for longer times. These data offer a mechanistic explanation of the protection of immobilized microorganisms from phenolic solvents. The data point to the membrane as an important cell component in the toxicity of these pollutants.     

Influence of phenols on growth and membrane permeability of free and immobilized Escherichia coli

The inhibition of the exponential growth of Escherichia coli K-12 by different phenolic compounds was examined. Cells entrapped in calcium alginate showed a greater tolerance than cells grown in suspension. The extent of inhibition of growth of the immobilized cells depended on the period of growth in the gel matrix. After the addition of bacteriostatic concentrations of phenol or 4-chlorophenol, a dose-dependent efflux of metabolites such as ATP and of K+ ions was elicited. Provided that glucose was supplied as an energy substrate, a reaccumulation of K+ ions at low phenol concentrations was observed. The restoration of the membrane gradient for K+ always preceded the continuation of growth in the presence of the toxic compounds. Compared with free cells, those cells immobilized and grown in alginate suffered a smaller loss of cations after the addition of 4-chlorophenol. The reestablishment of gradients was observed at higher concentrations of the pollutants with entrapped cells than with free cells. Corresponding to the increase in tolerance, the membrane damage was reduced in cells grown in immobilized form for longer times. These data offer a mechanistic explanation of the protection of immobilized microorganisms from phenolic solvents. The data point to the membrane as an important cell component in the toxicity of these pollutants.     

Detection of phenolic compounds using impedance spectroscopy measurements

Langmuir-Blodgett (LB) and layer-by-layer films (LbL) of a PPV (p-phenylenevinylene) derivative, an azo compound and tetrasulfonated phthalocyanines were successfully employed as transducers in an “electronic tongue” system for detecting trace levels of phenolic compounds in water. The choice of the materials was based on their distinct electrical natures, which enabled the array to establish a fingerprint of very similar liquids. Impedance spectroscopy measurements were taken in the frequency range from 10 Hz to 1 MHz, with the data analysed with principal component analysis (PCA). The sensing units were obtained from five-layer LB films of (poly[(2-methoxy-5-n-hexyloxy)-p-phenylenevinylene]), OC₁OC₁₈-PPV (poly(2-methoxy,5-(n-octadecyl)-p-phenylenevinylene)), DR (HEMA-co-DR13MA (poly-(hydroxyethylmethacrylate-co-[4′-[[2-(methacryloyloxy)-ethyl]ethylamino]-2-chloro-4-nitroazobenzene]))) and five-bilayer LbL films of tetrasulfonated metallic phthalocyanines deposited onto gold interdigitated electrodes. The sensors were immersed into phenol, 2-chloro-4-methoxyphenol, 2-chlorophenol and 3-chlorophenol (isomers) solutions at 1 × 10⁻⁹ mol L⁻¹, with control experiments carried out in ultra pure water. Samples could be distinguished if the principal component analysis (PCA) plots were made with capacitance values taken at 10³ Hz, which is promising for detection of trace amounts of phenolic pollutants in natural water.     

Continuous phenol biodegradation in a simple packed bed bioreactor of calcium alginate-immobilized <Emphasis Type="Italic">Candida tropicalis</Emphasis> (NCIM 3556)

Phenol biodegradation in a continuous system of immobilized Candida tropicalis NCIM 3556 was studied. The bioreactor was simple, it had a feed inlet from the bottom and the effluent outlet from top, no supplementary oxygen was supplied, the reactor was operated continuously for 116 days. Initially the column was run continuously with a feed concentration of 2 g l⁻¹ for 42 days whence a degradation of >97% was achieved. The feed concentration was then increased to 3 g l⁻¹, for which a ~80% biodegradation was sustained for 90 days after which there was a steady decrease in the performance. When the phenol degradation was reduced to ~50% in 116 days, the reactor was stopped. The efficiency of free cells recycled every 24 h and immobilized cells were compared; it was estimated that repeated reuse of free cells in batch mode gave an overall efficiency of 0.102 g phenol degradation g⁻¹ cell wet weight in 12 days. In contrast, the immobilized system of the same biomass had a longer working lifetime of ~4 months indicating an efficiency of 3.72 g phenol g⁻¹ cell wet wt.     

Enhanced phenol degradation by immobilized Acinetobacter sp. strain AQ5NOL 1

A locally isolated Acinetobacter sp. Strain AQ5NOL 1 was encapsulated in gellan gum and its ability to degrade phenol was compared with the free cells. Optimal phenol degradation was achieved at gellan gum concentration of 0.75% (w/v), bead size of 3 mm diameter (estimated surface area of 28.26 mm²) and bead number of 300 per 100 ml medium. At phenol concentration of 100 mg l⁻¹, both free and immobilized bacteria exhibited similar rates of phenol degradation but at higher phenol concentrations, the immobilized bacteria exhibited a higher rate of degradation of phenol. The immobilized cells completely degrade phenol within 108, 216 and 240 h at 1,100, 1,500 and 1,900 mg l⁻¹ phenol, respectively, whereas free cells took 240 h to completely degrade phenol at 1,100 mg l⁻¹. However, the free cells were unable to completely degrade phenol at higher concentrations. Overall, the rates of phenol degradation by both immobilized and free bacteria decreased gradually as the phenol concentration was increased. The immobilized cells showed no loss in phenol degrading activity after being used repeatedly for 45 cycles of 18 h cycle. However, phenol degrading activity of the immobilized bacteria experienced 10 and 38% losses after the 46 and 47th cycles, respectively. The study has shown an increased efficiency of phenol degradation when the cells are encapsulated in gellan gum.     

Biodegradation of Carbofuran phenol by free and immobilized cells of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> ATCC13883T

The Klebsiella sp. strain ATCC13883T capable of degrading carbofuran phenol (2,3-dihydro-2,2-dimethylbenzofuran-7-ol) has been separated from the soil by enrichment culture technique and immobilized in various, namely polyurethane foam (PUF), polyacrylamide, alginate, agar and alginate-bentonite clay-powdered activated charcoal (PAC). The degradation rates of 20 and 30 mM carbofuran phenol by free and immobilized cells in batch and semi-continuous shaken cultures were compared. The PUF-immobilized cells achieved higher degradation rates in a shorter time than freely suspended cells and the cells immobilized in polyacrylamide, alginate and agar. The PUF- and alginate-bentonite clay-PAC-immobilized cells could be reused for more than 36 cycles, polyacrylamide-entrapped cells for 20 cycles and alginate-bentonite-PAC 28 cycles, without losing any degradation capacity and showed better tolerance to pH, temperature and concentration changes than free cells. These results showed that cells immobilized in modified alginate-bentonite-PAC immobilizers tolerated and completely degraded carbofuran phenol at initial concentrations of 20 and 30 mM and also higher. Such a bacterial strain could be used for bioremediation of environments contaminated with phenolic compounds.     

Solid-phase extraction method for the determination of free and conjugated phenol compounds in human urine

A rapid flow system for automatic sample conditioning for the determination of phenol compounds in human urine has been developed and optimised. Free phenols are detected directly in urine samples while total phenols require acid hydrolysis to convert their conjugate fraction into free phenols, all compounds then being cleaned up and preconcentrated by solid-phase extraction. Separation and determination are done by gas chromatography, using mass spectrometry operating in the selective ion monitoring mode for quantitation. The linear range was 1-160 ng/ml of urine for most of the phenols. Limits of detection for phenol compounds (phenol, alkylphenols and chlorophenols) in the nanogram-per-millilitre range (0.3-0.6 ng/ml) are thus achieved by using 1 ml of urine; also, the repeatability, as RSD, is less than 6.5%. Based on the results for urine samples from unexposed individuals, 2-methylphenol, 2-chlorophenol and 2,4-dichlorophenol are largely detected in hydrolysed urine samples, whereas phenol and 4-methylphenol are detected in hydrolysed and unhydrolysed urine. Other chlorophenols such as trichlorophenols and pentachlorophenol are not detected. The results obtained in the analysis of urine from an individual before and after dietary intake reveal that the levels of phenol compounds in urine look related to food intake.     

Biodegradation of phenolic compounds by sulfate-reducing bacteria from contaminated sediments

The biodegradation of phenolic compounds under sulfate-reducing conditions was studied in sediments from northern Indiana. Phenol, p-cresol and 4-chlorophenol were selected as test substrates and added to sediment suspensions from four sites at an initial concentration of 10 mg/liter. Degradative abilities of the sediment microorganisms from the four sites could be related to previous exposure to phenolic pollution. Time to onset of biodegradation of p-cresol and phenol in sediment suspensions from a nonindustrialized site was approximately 70 and 100 days, respectively, in unacclimated cultures. In sediment slurries from three sites with a history of wastewater discharges containing phenolics, time to onset of biodegradation was 50–70 days for p-cresol and 50–70 days for phenol in unacclimated cultures. In acclimated cultures from all four sites, the length of the lag phase was reduced to 14–35 days for p-cresol and 25–60 days for phenol. Length of the biodegradative phase varied from 25 to 40 days for phenol and 10 to 50 days for p-cresol and was not markedly affected by acclimation. Substrate mineralization by sulfate-reducing bacteria was confirmed with radiotracer techniques using an acclimated sediment culture from one site. Addition of molybdate, a specific inhibitor of sulfate reduction, and bacterial cell inactivation inhibited sulfate reduction and substrate utilization. None of the sites exhibited the ability to degrade 4-chlorophenol, nor were acclimated phenol and p-cresol degrading cultures from a particular site able to cometabolize 4-chlorophenol.Correspondence to: D. Dean-Ross     

Impact of phenolic compounds on hydrothermal oxidation of cellulose

The effect of phenolic compounds on hydrothermal oxidation of cellulose was studied using a batch reactor at 300 degrees C with H(2)O(2) as oxidant. Intermediate products, as well as the yields of acetic acid produced in the oxidation of cellulose, phenolic compounds, and cellulose-phenolic compound mixtures were examined. Phenolic compounds used were phenol, 1,4-benzenediol, 2-methoxy-4-methylphenol, and 2,6-di-tert-butyl-4-methylphenol. In the case of oxidation of cellulose-phenolic compound mixtures, (1) formic acid, a basic oxidation product from carbohydrates, decreased considerably, (2) 5-hydroxymethyl-2-furaldehyde and 2-furaldehyde, acid-catalyzed dehydration products from carbohydrates, appeared, and (3) the yield of acetic acid increased compared to that in the oxidation of cellulose. From these results, phenolic compounds seem to inhibit the oxidation of cellulose under hydrothermal conditions. The inhibition of the oxidation of cellulose by phenolic compounds seems to be related closer to the stability of phenolic compounds under oxidation conditions rather than the ease to remove phenolic hydrogen on the OH group.     

Biodegradation of phenol by immobilized Aspergillus awamori NRRL 3112 on modified polyacrylonitrile membrane

Covalent immobilization of Aspergillus awamori NRRL 3112 was conducted onto modified polyacrylonitrile membrane with glutaraldehyde as a coupling agent. The polymer carrier was preliminarily modified in an aqueous solution of NaOH and 1,2-diaminoethane. The content of amino groups was determined to be 0.58 mgeq g⁻¹. Two ways of immobilization were used—in the presence of 0.2 g l⁻¹ phenol and without phenol. The capability of two immobilized system to degrade phenol (concentration—0.5 g l⁻¹) as a sole carbon and energy source was investigated in batch experiments. Seven cycles of phenol biodegradation were conducted. Better results were obtained with the immobilized system prepared in the presence of phenol, regarding degradation time and phenol biodegradation rate. Scanning electron micrographs of the polyacrylonitrile membrane/immobilized Aspergillus awamori NRRL at the beginning of repeated batch cultivation and after the 7th cycle were compared. After the 7th cycle of cultivation the observations showed large groups of cells. The results from the batch experiments with immobilized system were compared to the results produced by the free strain. Phenol biodegradation experiments were carried out also in a bioreactor with spirally wound membrane with bound Aspergillus awamori NRRL 3112 in a regime of recirculation. 10 cycles of 0.5 g l⁻¹ phenol biodegradation were run consecutively to determine the degradation time and rate for each cycle. The design of the bioreactor appeared to be quite effective, providing large membrane surface to bind the strain.     

Immobilization of laccase from Streptomyces psammoticus and its application in phenol removal using packed bed reactor

Laccase was produced from Streptomyces psammoticus under solid-state fermentation. The enzyme was partially purified by ammonium sulphate precipitation and was immobilized in alginate beads by entrapment method. Calcium alginate beads retained 42.5% laccase activity, while copper alginate beads proved a better support for laccase immobilization by retaining 61% of the activity. Phenol and colour removal from a phenol model solution was carried out using immobilized laccase. Batch experiments were performed using packed bed bioreactor, containing immobilized beads. Reusability of the immobilized matrix was studied for up to 8 successive runs, each run with duration of 6 h. The system removed 72% of the colour and 69.9% of total phenolics from the phenol model solution after the initial run. The immobilized system maintained 50% of its efficiency after eight successive runs. The degradation of phenolic compounds by immobilized laccase was evaluated and confirmed by Thin layer chromatography and nuclear magnetic resonance spectroscopy.     

Computer calculation-based quantitative structure-activity relationships for the oxidation of phenol derivatives horseradish peroxidase compound II

 The second-order rate constants for the oxidation of a series of phenol derivatives by horseradish peroxidase compound II were compared to computer-calculated chemical parameters characteristic for this reaction step. The phenol derivatives studied were phenol, 4-chlorophenol, 3-hydroxyphenol, 3-methylphenol, 4-methylphenol, 4-hydroxybenzoate, 4-methoxyphenol and 4-hydroxybenzaldehyde. Assuming a reaction of the phenolic substrates in their non-dissociated, uncharged forms, clear correlations (r = 0.977 and r = 0.905) were obtained between the natural logarithm of the second-order rate constants (ln k _app and ln k ₂ respectively) for their oxidation by compound II and their calculated ionisation potential, i.e. minus the energy of their highest occupied molecular orbital [E(HOMO)]. In addition to this first approach in which the quantitative structure-activity relationship (QSAR) was based on a calculated frontier orbital parameter of the substrate, in a second and third approach the relative heat of formation (ΔΔHF) calculated for the process of one-electron abstraction and H^• abstraction from the phenol derivatives was used as a parameter. Plots of the natural logarithms of the second-order rate constants (k _app and k ₂) for the reaction and the calculated ΔΔHF values for the process of one-electron abstraction also provide clear QSARs with correlation coefficients of –0.968 and –0.926 respectively. Plots of the natural logarithms of the second-order rate constants (k _app and k ₂) for the reaction and the calculated ΔΔHF values for the process of H^• abstraction provide QSARs with correlation coefficients of –0.989 and –0.922 respectively. Since both mechanisms considered, i.e. initial electron abstraction versus initial H^• abstraction, provided clear QSARs, the results could not be used to discriminate between these two possible mechanisms for phenol oxidation by horseradish peroxidase compound II. The computer calculation-based QSARs thus obtained for the oxidation of the various phenol derivatives by compound II from horseradish peroxidase indicate the validity of the approaches investigated, i.e. both the frontier orbital approach and the approach in which the process is described by calculated relative heats of formation. The results also indicate that outcomes from computer calculations on relatively unrelated phenol derivatives can be reliably compared to one another. Furthermore, as the actual oxidation of peroxidase substrates by compound II is known to be the rate-limiting step in the overall catalysis by horseradish peroxidase, the QSARs of the present study may have implications for the differences in the overall rate of substrate oxidation of the phenol derivatives by horseradish peroxidase. Received: 29 March 1996 / Accepted: 17 July 1996     

Reducing effect of aerobic selector on the toxicity of synthetic organic compounds in activated sludge process

The effects of phenol, 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP) and 1,2,4-trichlorobenzene (1,2,4-TCB) on the biodegradation kinetics of the conventional activated sludge system (CASS) and the selector activated sludge system (SASS) were investigated. Experiments were carried out using a respirometric method on unacclimated biomass from two lab-scale systems that were operated with the sludge age of 8 days. Toxicity of the test compounds for both reactors were arranged according to EC₅₀ (effective concentration) values in order as: 1,2,4-TCB > 2,4-DCP > 2-CP > phenol. All selected test compounds induced higher inhibition effect in the CASS. The SASS appeared to reduce inhibition effect in comparison to the CASS, by 21.36%, 66.95%, 64.37% and 33.33% for phenol, 2-CP, 2,4-DCP and 1,2,4-TCB, respectively. Consequently, the SASS may be recommended as a promising configuration alternative for the waste streams containing toxic compounds.     

Recent advances in the development of biosensor for phenol: a review

Phenol and its derivatives are widespread contaminants whose sources are both natural and industrial. Phenol is massively produced and used as a starting material for synthetic polymers and fibers. Although phenolic compounds play important biochemical and physiological roles in living systems, their accumulation in the environment as a result of intensive human activity may result in drastic ecological problem. Various analytical techniques are available for the detection of phenol in environmental samples. But they need complex sample pre-treatment so as are time consuming, costly and use heavy devices. On the other hand a biosensor is a device that gives rapid detection, cost effective and easy. A review study was carried out to accumulate the possible biosensors for the detection of phenolic compounds in environmental samples. A number of biological components including microorganisms, enzymes, antibodies, antigens, nucleic acids etc. can be used for the construction of biosensors that was found to detect phenolic compounds. Of all type of biological components microorganisms and enzymes are mostly used. The microorganisms are Pseudomonas, Moraxella, Arthrobacter, Rhodococcus, and Trichosporon. The most used enzymes are tyrosinase, peroxidase, laccase, glucose dehydrogenase, cellobiose dehydrogenase etc. Antibody sensors can detect a very trace level. The biorecognition of DNA biosensors occur by hybridization of DNA. Biosensors are found to work well when the biological sensing element is immobilized. A variety of immobilization techniques were found to use as adsorption, covalent binding, entrapment, cross-linking etc. For immobilization the matrices used was polyvinyl alcohol, Osmium complex, nafion/sol?Cgel silicate, chitosan, silica gel etc.     

Inhibition studies of bacterial α-carbonic anhydrases with phenols

The α-class carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogens Neisseria gonorrhoeae (NgCAα) and Vibrio cholerae (VchCAα) were investigated for their inhibition by a panel of phenols and phenolic acids. Mono-, di- and tri-substituted phenols incorporating additional hydroxyl/hydroxymethyl, amino, acetamido, carboxyl, halogeno and carboxyethenyl moieties were included in the study. The best NgCAα inhibitrs were phenol, 3-aminophenol, 4-hydroxy-benzylalcohol, 3-amino-4-chlorophenol and paracetamol, with K_I values of 0.6–1.7 µM. The most effective VchCAα inhibitrs were phenol, 3-amino-4-chlorophenol and 4-hydroxy-benzyl-alcohol, with K_I values of 0.7–1.2 µM. Small changes in the phenol scaffold led to drastic effects on the bacterial CA inhibitory activity. This class of underinvestigated bacterial CA inhibitors may thus lead to effective compounds for fighting drug resistant bacteria.     

Kinetics of the biodegradation of phenol in wastewaters from the chemical industry by covalently immobilized <Emphasis Type="Italic">Trichosporon cutaneum</Emphasis> cells

A simple method for the preparation of the biocatalyst with whole cells is presented, and the applicability of the technique for biodegradation of phenol in wastewater from the chemical industries using the basidomycetes yeast Trichosporon cutaneum is explored. Kinetic studies of the influence of other compounds contained in wastewater as naphthalene, benzene, toluene and pyridine indicate that apart from oil fraction, which is removed, the phenol concentration is the only major factor limiting the growth of immobilized cells. Mathematical models are applied to describe the kinetic behavior of immobilized yeast cells. From the analysis of the experimental curves was shown that the obtained values for the apparent rate parameters vary depending on the substrate concentration (μ_maxapp from 0.35 to 0.09 h⁻¹ and K _sapp from 0.037 to 0.4 g dm⁻³). The inhibitory effect of the phenol on the obtained yield coefficients was investigated too. It has been shown that covalent immobilization of T. cutaneum whole cells to plastic carrier beads is possible, and that cell viability and phenol degrading activity are maintained after the chemical modification of cell walls during the binding procedure. The results obtained indicate a possible future application of immobilized T. cutaneum for destroying phenol in industrial wastewaters.