Zlatka Alexieva

Microbial degradation of phenol and phenolic derivatives

Phenol and its derivatives are one of the largest groups of environmental pollutants due to their presence in many industrial effluents and broad application as antibacterial and antifungal agents. A number of microbial species possess enzyme systems that are applicable for the decomposition of various aliphatic and aromatic toxic compounds. Intensive efforts to screen species with high‐degradation activity are needed to study their capabilities of degrading phenol and phenolic derivatives. Most of the current research has been directed at the isolation and study of microbial species of potential ecological significance. In this review, some of the best achievements in degrading phenolic compounds by bacteria and yeasts are presented, which draws attention to the high efficiency of strains of Pseudomonas, Candida tropicalis, Trichosporon cutaneum, etc. The unique ability of fungi to maintain their degradation potential under conditions unfavorable for other microorganisms is outstanding. Mathematical models of the microbial biodegradation dynamics of single and mixed aromatic compounds, which direct to the benefit of the processes studied in optimization of modern environmental biotechnology are also presented.

Biodegradation of toxic organic components from industrial phenol production waste waters by free and immobilized Trichosporon cutaneum R57

Abstract The ability of Trichosporon cutaneum R57 to grow on and utilize some toxic compounds (phenol, acetophenone, acetone, α-methylstyrene, benzoic acid, dimethyl phenyl carbinol, methanol and isopropylbenzene) as sole carbon and energy sources was studied. The growth of T. cutaneum R57 in a model solution including all the contaminants mentioned above was studied and results were compared with growth curves in two types of waste waters taken from the basin of the biopurification station with active sludge, treating waters from an oil refinery and some accompanying organic products facilities, including waste water from industrial phenol production. The strain was found to grow well in both types of waste waters (inlet and outlet of the biobasin) and in the model solution. The results showed that strain T. cutaneum R57 can utilize a wide range of toxic compounds simultaneously. The growth of free and immobilized cells in the waste waters from the phenol production facility after an anion-exchange (Amberlite HAD-4 resin) purification stage was also studied. The immobilized system had high vitality and adaptivity into waste water from phenol production. The highest degradation after 24 h was observed for acetone (50–60%), benzoic acid was reduced by 25–40% and phenol and dimethyl phenyl carbinol approximately by 30–45% depending on the type of the cells. Immobilized cells showed a higher ability to degrade the more toxic compounds compared to free cells. The results obtained indicate the ability of T. cutaneum R57 to degrade some of the most toxic contaminants present in waste waters.

Phenol and cresol mixture degradation by the yeast Trichosporon cutaneum

Most industrial wastes contain different organic mixtures, making important the investigation on the microbial destruction of composite substrates. The capability of microbes to remove harmful chemicals from polluted environments strongly depends on the presence of other carbon and energy substrates. The effect of mixtures of phenol- and methyl-substituted phenols (o-, m-, p-cresol) on the growth behaviour and degradation capacity of Trichosporon cutaneum strain was investigated. The cell-free supernatants were analysed by HPLC. It was established that the presence of o-, m- and p- cresol has not prevented complete phenol assimilation but had significant delaying effect on the phenol degradation dynamics. The mutual influence of phenol and p-cresol was investigated. We developed the kinetic model on the basis of Haldane kinetics, which used model parameters from single-substrate experiments to predict the outcome of the two-substrate mixture experiment. The interaction coefficients indicating the degree to which phenol affects the biodegradation of p-cresol and vice versa were estimated. Quantitative estimation of interaction parameters is essential to facilitate the application of single or mixed cultures to the bio-treatment of hazardous compounds.

Decolorization of Synthetic Dye Reactive Blue 4 by Mycelial Culture of White-Rot Fungi Trametes Versicolor 1

ABSTRACT The industry is a major source of pollution for water ecosystems. Industrial production of textile, cellulose and various chemicals is connected with synthetic dyes usage. The discharged effluents could have a hazardous influence on the environment. The biological treatment for synthetic dyes removal is a very perspective, environmentally protective and low cost approach for solution of such problems. One of the often used and very important in dyeing of cellulosic fabrics and textile industry dyes is the anthraquinone-base chlorotriazine dye, known as Reactive Blue 4. Decolorization of Reactive Blue 4 by Trametes versicolor strain 1 was investigated. The experiments were carried out with different concentrations of dye (50mg/l and 125mg/l) and glucose (1, 2 and 3%) in a medium. The enzyme activity of laccase (EC 1.10.3.2) was measured during the process of decolorization. It was shown that there was a direct correlation between the observed enzyme activity and the investigated process effectiveness. It was established that the best conditions for laccase production and decolorization of 125mg/l Reactive Blue 4 dye are in a medium containing 3% glucose. In these conditions 90% Reactive Blue 4 was decolorized for 384 hours.

Phenol utilization by filamentous yeast Trichosporon cutaneum

Abstract The investigated strain Trichosporon cutaneum shows well expressed capability for metabolizing high concentrations of phenol, up to 1 g/l, utilizing it as the sole carbon source for the growth and development of the population. The data reported, prove the good perspectives for its application in protecting the environment from phenol pollution. No data about modelling the process of cultivation of Trichosporon cutaneum in phenol media is available in scientific literature up to now. The mathematical model, reported here, consists of two nonlinear differential equations, describing cell growth and substrate consumption. The unknown parameters are estimated following the method of Hooke and Jeeves. A number of simulation investigations are carried out. They prove the adequacy of the model and its applicability in further studies on the processes of growth and phenol uptake of Trichosporon cutaneum.

Kinetic Parameters Determination of the Phenolic Derivatives Assimilation by Trichosporon Cutaneum R57

ABSTRACT The kinetic parameters characteristic of the growth and degradation capacity of Trichosporon cutaneum R57 strain in relation to o-, m-, p-chlorophenol, o-, m-, p-cresol and o-, m-, p-nitrophenol are presented in this study. The investigated strain degraded up to 0.1 g/l of all mono-chlorophenols. A concentration of 0.2 g/l p- cresol was completely degraded whereas m- cresol was degraded to an extent of 32%. No degradation of o- cresol was observed. O- nitrophenol in concentration of 0.1 g/l was less readily degraded than the same concentration of m-nitrophenol and p- nitrophenol was not degraded. The Haldane equation has frequently been used to describe the biodegradation of toxic substrates but little is known about the kinetic constants for the biodegradation of aromatic compounds. The coefficients obtained confirmed the higher toxicity of the investigated phenol derivatives on Trichosporon cutaneum R57. The yield coefficient (Y) was least of all in the medium supplemented with o- chlorophenol where μmax was also minimum. The highest value of Ki characteristic of p-cresol assimilation correlated with the ability of Trichosporon cutaneum R57 to degrade up to 0.4 g/l of this compound. The model prediction was compared with experimental data. It was found that the designed model described the trend of experimental data satisfactorily.

Influence of various phenolic compounds on phenol hydroxylase activity of a Trichosporon cutaneum strain.

The phenol-degrading strain Trichosporon cutaneum R57 utilizes various aromatic and aliphatic compounds as a sole carbon and energy source. The intracellular activities of phenol hydroxylase [EC 1.14.13.7] of a Trichosporon cutaneum R57 strain grown on phenol (0.5 g/l) were measured. Different toxic phenol derivatives (cresols, nitrophenols and hydroxyphenols) were used as substrates in the reaction mixture for determination of the enzyme activity. The data obtained showed that the investigated enzyme was capable to hydroxylate all applied aromatic substrates. The measured activities of phenol hydroxylase varied significantly depending on the aromatic compounds used as substrates. The rate of phenol hydroxylase activity with phenol as a substrate (1.0 U/mg total cell protein) was accepted as 100%.

Influence of phenolic substrates utilised by yeast Trichosporon cutaneum on the degradation kinetics

The degradation kinetics of different phenolic substrates utilised by Trichosporon cutaneum R57 was studied. The following compounds were used as substrates: phenol, resorcinol, hydroquinone, 3-nitrophenol, 2,6-dinitrophenol, 3-chloro phenol and p-cresol. The specific degradation rates (Qs) were described by a Haldane kinetic model. The unknown model parameters were estimated using the mathematical optimisation procedure for direct search. The results obtained demonstrated that Qs varied greatly in the experiments carried out. The level of biodegradability depended on the different structure and toxicity of compounds used as carbon substrates. The highest Qs values were observed for less toxic hydroxylated phenols (0.77–0.85 h−1), while the most toxic chlorinated phenols were characterised with the lowest Qs values (0.224 h−1). The results obtained with different concentrations of resorcinol (from 0.2 to 0.8 g L−1) and 2,6-dinitrophenol (from 0.2 to 0.7 g L−1) demonstrated a growing inhibitory effect directly correlating with the extended time necessary for complete degradation of both compounds.

Molecular Analysis of Phenol-Degrading Microbial Strains

In an attempt to estimate the occurrence of phenol hydroxylase-related gene sequences we performed a dot blot hybridization assay with DNA from phenol utilizing Trichosporon cutaneum R57 strain NBIMCC 2414 and microbial isolates from different wastewaters. The used oligonucletides were homologous to the 5 ′-end of TORPHD locus (NCBI)-coding phenol hydroxylase in Trichosporon cutaneum ATCC 46490 and to the 5 ′-end of TORCCMLE locus (NCBI)-coding cis,cis-muconate-lactonizing enzyme in Trichosporon cutaneum ATCC 58094. Two microbial strains, Escherichia coli JM 109 and Lactobacillus acidophilus ATCC 4356, incapable to degrade phenol were used as negative controls. We established the presence of hybridization with both used oligonucleotide probes in T. cutaneum R57 and T. cutaneum ATCC 46490 yeast strains. The experiments implemented with microbial isolates obtained from three industrialized areas in Bulgaria showed that 7 of them may carry sequences hybridizing with a phenol hydroxylase oligonucleotide probe. A subsequent hybridization test for the cis,cis-muconate-lactonizing enzyme showed that only 3 of them displayed a positive signal. Lactobacillus acidophilus ATCC 4356 and Escherichia coli JM 109 strains’ DNA used as negative controls in the experiments did not reveal any sequence similarity to the both applied oligonucleotides. The partial nucleotide sequences of 16S rDNAs of the isolated strains C1 and K1 obtained as PCR products were determined and sequenced. A comparison of these nucleotide sequences with similar sequences in NCBI Data Bank indicated that both C1 and K1 strains are closely related to the genera Acinetobacter and Burkholderia.

Decolorization of Industrial Dyes by Immobilized Mycelia of Trametes Versicolor

ABSTRACT The aim of this study was to compare the decolorization of several of the most utilized synthetic dyes in textile applications by immobilized white-rot fungus Trametes versicolor. Different immobilization methods for achieving maximum decolorization were explored. The initial dye concentration was 125 mg/L and the immobilized preparation concentration was 10% (w/v). Different degrees of decolorization were observed. Different operational stability of the immobilized preparations was accomplished. Increase of the immobilized preparation concentration up to 30% (w/v) was investigated. The study was performed in two stages: simulated incubation in a ‘batch’ reactor and trickle-bed continuous flow reactor During the decolorization process, laccase activity was detected. The dye-decolorizing activity of the immobilized culture was found to be associated with the processes of biodegradation, bio-oxidation and biosorption.