Maria Gerginova

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.

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.

The Effect of Cu2+ and Cd2+ on Citric Acid Production by Saccharomycopsis Lipolytica Strains

ABSTRACTThe influence of copper and cadmium on the microbial growth and production was assessed in conjunction with three Saccharomycopsis lipolytica strains, which are used particularly as citric acid producers. Stable metal resistant mutants were obtained. The main kinetic parameters specific growth rate and specific rate of product formation of the mutant strains were determined and compared to these of the wild type.