Cutaneum R57 Research Articles

Biodegradation and Utilization of the Pesticides Glyphosate and Carbofuran by Two Yeast Strains

The widespread use of synthetic pesticides in agricultural practice is associated with the risk of environmental pollution, damage to non-target organisms, and harm to the health of consumers due to the presence of pesticides in the resulting products. Metabolically active microbial species play a significant role in eliminating harmful chemicals from the environment. The two yeast strains used in this study are Trichosporon cutaneum R57 and Candida tropicalis V1. Both strains showed growth and development in the presence of the pesticides glyphosate and carbofuran used as the sole carbon sources. The gas-chromatographic analysis performed showed that C. tropicalis V1 reached 76% of 0.3 g/L glyphosate biodegradation in 192 h. At the same time, the amount of glyphosate decreased by about 58% in the culture of T. cutaneum R57. During the glyphosate biodegradation process, the presence of the intermediate metabolites methylglycine and glycine was revealed. Strain T. cutaneum R57 demonstrated nearly total biodegradation of 0.3 g/L carbofuran in 192 h of cultivation. Strain C. tropicalis V1 showed a slow decrease (23.4%) of the same amount of carbofuran during 192 h. Pyruvic acid and carbofuran-7-phenol have been found to be intermediate metabolites in the breakdown of carbofuran. This report provides new information on the potential of yeasts to participate in environmental pesticide cleanup processes.

The oxidative stress response of the filamentous yeast Trichosporon cutaneum R57 to copper, cadmium and chromium exposure

Despite the intensive research in the past decade on the microbial bioaccumulation of heavy metals, the significance of redox state for oxidative stress induction is not completely clarified. In the present study, we examined the effect of redox-active (copper and chromium) and redox-inactive (cadmium) metals on the changes in levels of oxidative stress biomarkers and antioxidant enzyme defence in Trichosporon cutaneum R57 cells. This filamentous yeast strain showed significant tolerance and bioaccumulation capability of heavy metals. Our findings indicated that the treatment by both redox-active and redox-inactive heavy metal induced oxidative stress events. Enhanced concentrations of Cu2+, Cr6+ and Cd2+ caused acceleration in the production of reactive oxygen species (ROS), increase in the level of oxidatively damaged proteins and accumulation of reserve carbohydrates (glycogen and trehalose). Cell response against heavy metal exposure also includes elevation in the activities of antioxidant enzymes, superoxide dismutase and catalase, which are key enzymes for directly scavenging of ROS. Despite the mentioned changes in the stress biomarkers, T. cutaneum did not show a significant growth diminution. Probably, activated antioxidant defence contributes to the yeast survival under conditions of heavy metal stress.

Synthesis and characterization of hybrid materials with embedded silver nanoparticles and their application as antimicrobial matrices for waste water purification

Polyvinyl alcohol/aminopropyltriethoxysilane hybrid materials with embedded silver nanoparticles (PVA/APTEOS/AgNps) have been synthesized for the first time using sol–gel method. The successful formation of silver nanoparticles embedded into PVA/APTEOS matrices has been proven by UV–vis and TEM analyses. The presence of fungicidal activity of thus prepared PVA/APTEOS/AgNps materials against different control and clinical strains has been established applying disk diffusion method (DDM). The hybrid materials were tested as matrices for immobilization of Trichosporon cutaneum R57 capable to remove Mn2+ from aqueous solutions. This strain showed considerable ability to remove Mn2+ ions from aqueous solutions at low silver nanoparticles content into the PVA/APTEOS matrices.

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.

PVA -based Hybrid Materials for Immobilization of Trichosporon cutaneum R57 Efficient in Removal of Chromium Ions

New hybrid materials based on polyvinyl alcohol (PVA) and γ-aminopropyltriethoxyxilane (APTEOS), 3-mercaptopropyltriethoxysilane (MPTEOS) and tetraethoxysilane (TEOS) were prepared. To determine the thermal stability of the hybrid materials, thermogravimetric analysis (TGA) was performed. The new materials were tested for biofilm formation and biosorption studies. They were tested as matrices for immobilization of Trichosporon cutaneum R57 capable to remove Cr(VI) from aqueous solutions. The use of APTEOS and MPTEOS as precursors, incorporated new NH– and SH– bonds, which ensured additional binding places for cells immobilization. The presence of SH– and NH– groups in the hybrid matrices resulted in apparently higher Cr(VI) sorption capacity of the immobilized Tr. cutaneum R57 cells due to the incorporation of additional adsorption sites in the matrix through the PVA functionalities. The synthesized PVA/APTEOS materials proved efficient for use in biosorption applications.

Immobilization of Trichosporon Cutaneum R57 on PVA/TEOS/MPTEOS Hybrid Matrices for Removal of Manganese Ions

ABSTRACTNew hybrid materials based on polyvinyl alcohol (PVA), 3-mercaptopropyltriethoxysilane (MPTEOS) and tetraethoxysilane (TEOS) were prepared, using a sol-gel process. Thermogravimetric analysis (TGA) was performed to determine the thermal stability of the hybrid materials. MPTEOS, as a precursor, ensured the presence of SH-groups, thus providing additional binding places for cell immobilization. Based on good adhesive behavior, the hybrid materials were tested as matrices for immobilization of Trichosporon cutaneum R57 for removal of manganese ions from aqueous solutions. Maximum Mn2+ removal was achieved by cells immobilized onto materials with higher MPTEOS concentration. The synthesized PVA/TEOS/MPTEOS hybrid materials proved to be efficient for use in biosorption applications.

Biodegradation of Phenol and Phenolic Derivatives by a Mixture of Immobilized Cells of Aspergillus Awamori and Trichosporon Cutaneum

ABSTRACTTwo immobilized systems of Aspergillus awamori NRRL3112 and Trichosporon cutaneum R57 on modified polyamide beads were obtained. The optimal conditions for immobilization of strains Aspergillus awamori NRRL3112 and Trichosporon cutaneum R57 on polyamide beads were determined. It was found that by combined action of two immobilized systems it is possible to degrade up to 1.5 g·L−1 phenol, while individually immobilized and free cells did not have this capacity (degrade up to 1 g·L1). The high potential possibility and the synergistic action of both immobilized systems were proved. Biodegradation of different phenolic derivatives was investigated - 2\\-chlorophenol (2-CP), 3-chlorophenol (3-CP), 4-chlorophenol (4-CP), 2,4-dichlorophenol (2,4-DCP), 4-n-nonylphenol (4-NP), 3-methoxyphenol (3-MP), bisphenol A (BPA), 4-chloro-3,5-dimethylphenol (chloroxylenol—ChX), and 2-benzyl-4-chlorophenol (chlorophen—ChP). It was found that the utilization of phenol, BPA, and 2,4-DCP was very fast (16 h); 2-CP, 3-CP, 4-CP, and 3-MP were utilized for 40 h in the range from 44 % to 72 %; NP and ChP were degraded very slowly (about 30 % for 40 h), while ChX remained unchanged for 40 h. The studied phenolic derivatives were listed in the following order, depending on the degree of biodegradation by the two immobilized strains Aspergillus awamori NRRL3112 and Trichosporon cutaneum R57: Phenol > BPA > 2,4-DCP > 3-MP > 3-CP > 2-CP > 4-CP > ChP > NP > ChX. The rate of biodegradation of phenol and some phenolic derivatives with a concentration of 0.1 g·L−1 and 0.3 g·L−1 were determined. The advantages of the two combined immobilized systems for biodegradation of phenol and phenolic derivatives as compared to the separate immobilized systems of the two stains and free cells were proved.

Removal of cadmium and copper ions by Trichosporon cutaneum R57 cells immobilized onto polyvinyl alcohol/tetraethoxysilane hybrid matrices

Abstract Polyvinyl alcohol (PVA) and tetraethoxysilane (TEOS) hybrid materials were prepared by sol-gel methods and tested as matrices for immobilization of Trichosporon cutaneum R57, capable of removing cadmium and copper ions from aqueous solutions. A kinetic model was applied and the effects of matrix TEOS content on the copper and cadmium uptake equilibria and rate constants were investigated.

Biobleaching of Flax Fibers by Degradation of Lignin with Phanerochaete chrysosporium and Trichosporon cutaneum R57

ABSTRACT The possible applications of lignin degrading enzymes obtained through the cultivation of a Trichosporon cutaneum R57 strain and white rot fungus Phanerochaete chrysosporium in flax fiber treatment was studied. The lightness of enzymatically processed fibers is significantly improved while the quantity of nondegraded lignin is lower. It has been found out that the hydrogen peroxide additionally applied to the enzymatic treatment strongly influences these effects.

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.

Monitoring of aromatic pollutants biodegradation

Trichosporon cutaneum R57 is known as effective biodegradant able to utilize and thus remove a number of toxic aromatic compounds from the environment. In the present study, the dynamics of the processes of degradation of monohydoxyl derivatives of phenol (resorcinol, catechol and hydroquinone) in concentrations up to 1.6 g/l were investigated and inhibitory constants ( k i) of these compounds were determined 0.58, 0.55, and 0.6 g/l correspondingly. The biodegradation of significantly more toxic aromatic compounds 2,6-dinitrophenol ( k i = 0.13 g/l), α-methylstyrene ( k i = 0.25 g/l) and acetophenone ( k i = 0.15 g/l) was also described. Based on summarized qualitative and quantitative data and the “if-then” rules developed, a fuzzy model was designed to describe the processes despite the degree of toxicity and concentration of the aromatic compounds. The output linguistic variables “ normal”, “ check” and “ attention” were defined, which determine further measures for improvement of process quality. The model suggested provides opportunity for early estimation of the biodegradation process quality.

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%.

Biodegradation of phenol by immobilized Trichosporon cutaneum R57 on modified polymer membranes

Covalent immobilization of Trichosporon cutaneum R57 cells was conducted onto PA and PAN membranes with glutaraldehyde as a coupling agent. Amount of immobilized cells was determined: for polyamide—1.4 mg cm −2 and for polyacrylonitrile—1.8 mg cm −2 (dry weight). The efficiency of each immobilized system was evaluated by the extent of phenol biodegradation (1.0 g l −1). Polyacrylonitrile-immobilized system completed the process for 45 h, whereas polyamide-immobilized system for 51 h. During the six next cycles the complete biodegradation time for polyacrylonitrile-immobilized system was reduced at 24, 18 and 15 h for III, V, VII cycles, respectively. The biodegradation time at eighth cycle was prolonged (42 h) and this is probably due to substrate diffusion limitations to the local accumulation of microbial cells on the membrane surface and cause cell death. Comparing the SEM of immobilized microbial cells on polyacrylonitrile membrane before the first cycle and after the eight cycle could be seen that great amount of immobilized cells are accumulated on the membrane surface during the seven biodegradation cycles.

Selection and Characterization of ʟ-Ethionine Resistant Mutants of Trichosporon cutaneum

Trichosporon cutaneum R57 and its L-ethionine resistant mutant NZ94 strain were investigated. The amino acid analyses of cell content of both strains were carried out. The pool of free methionine in the mutant strain is enhanced 16.5 times. The total amount of sulphur-containing amino acids in the mutant cells was significantly increased from 36.8 in the wild strain to 113.4 mg/g protein in the mutant strain. In the process of mutant strain cultivation there was found a high excretion of free methionine (259 microg/ml) in the medium. It was shown that the amino acid content of both wild and mutant strains would be helpful for formulating of new improved animal nutritional diets.

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

ABSTRACTThe 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.

Comparison of growth kinetics and phenol metabolizing enzymes of Trichosporon cutaneum R57 and mutants with modified degradation abilities

The model kinetic coefficients of the growth and phenol degradation processes of Trichosporon cutaneum R57 strain and the investigated mutants in culture media containing phenol as a sole carbon and energy source are reported. Successfully simulated phenol degradation profiles in all studied strains were obtained by Haldane-type kinetics. The data presented in the paper showed no direct correlation between efficiency of phenol degradation by the strains studied and biomass productivity. The observed significant increase of inhibitory coefficients in both 2R ( K i=0.46 g l −1) and 4R ( K i=0.44 g l −1) mutants corresponded with their improved capacity to degrade phenol and increased phenol hydroxylase activity. The intracellular specific activities of phenol hydroxylase (EC 1.14.13.7) and catechol 1,2-dioxygenase (EC 1.13.11.1) in T. cutaneum R57 strain and its mutants are measured. The comparison of data for phenol hydroxylase and catechol 1,2-dioxygenase activities in cell-free extracts obtained by ultrasonication and in permeabilized cells showed that the method of cell permeabilization was more favorable for enzyme analyses. The obtained specific activity of phenol hydroxylase in T. cutaneum R57 was 0.8 U mg −1 protein while those in 2R and 4R mutant strains were 1.47 U mg −1 protein and 1.28 U mg −1 protein, respectively. The value of catechol 1,2-dioxygenase activity showed quite less variability and was kept about 0.2 U mg −1 protein in all investigated strains. The results received in these experiments demonstrated that the increased rate of the initial phenol degradation reaction enhanced phenol utilization by cells and vice versa.

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

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.

Degradation of some phenol derivatives by Trichosporon cutaneum R57

The purpose of the present study was to determine whether Trichosporon cutaneum R57 was able to grow and utilize some phenol derivatives: resorcinol, 2,6-dinitrophenol, 3-nitrophenol, 4-nitrophenol and m-cresol as sole carbon and energy source. The results obtained showed the ability of the strain to develop by degrading these compounds, except 4-nitrophenol. Resorcinol and 2,6-dinitrophenol were utilized in 27 h in yeast nitrogen base (YNB) medium while 3-nitrophenol and m-cresol were only partly degraded in this period of time. The growth of T. cutaneum using YNB containing one of the phenol derivatives as the sole carbon and energy source was compared with the growth of cells in YNB with the same concentration of the phenol derivative plus 0.3 g l −1 phenol. Both compounds were simultaneously utilized by the mechanism of cometabolic degradation. HPLC analysis of the centrifuged culture medium showed an increased degradation rate of resorcinol. Resorcinol and phenol were completely degraded in 18 h. No significant change of the degradation rate for 2,6-dinitrophenol and 3-nitrophenol was observed. The combination of m-cresol and phenol exhibited inhibiting effects with negligible degradation of phenol and m-cresol in 27 h. The biodegradation of the phenolic derivatives by cells of T. cutaneum R57 immobilized onto polyamide and polyacrylonitrile granules and fibers was also studied. The results were similar to these obtained from free cells, although the period of biodegradation was found to be by ca. 3 h longer.

Cell immobilization of Trichosporon cutaneum strain with phenol degradation ability on new modified polymer carriers

Chemical immobilization of Trichosporon cutaneum R57 cells onto modified polyacrylonitrile granules and fibres was carried out. The polymer carriers were modified in water solutions of NaOH and 1,2-diaminoethane. The concentration of amino groups obtained by the modification was determined. The optimal conditions for chemical immobilization of cells of T. cutaneum R57 with glutaraldehyde were found to be: 40% NaOH concentration, 46.8 mg/ml initial cell suspension, 24 h immobilization time and pH 6. The change in protein concentration of free and immobilized cells was studied during a full biodegradation cycle. The rate of phenol biodegradation by T. cutaneum R57 cells immobilized on porous polyacrylonitrile (PAN) fibres was higher than that obtained using modified PAN beds.

Regulation of the First Two Enzimes of Methionine Biosynthesis in Trichosporon Cutaneum

ABSTRACTTrichosporon cutaneum R57 and it s derivative strain NZ94 are able to accumulale significant cellular amounts of methionine. Comparative analysis of the function and regulation of aspartate kinase and homoserine dehydrogenase in both strains was carried out. The activities of the first enzyme in the parental strain appeared to be regulated through repression and retroinhibition by treonine molecules in the culture medium. In the mutant strain NZ 94 the aspartate kinase activity was 33% higher compared to this of R57 and was resistant to threonine inhibition. The activity of homoserine dehidrogenase in both strains showed equal values and were repressed and retroinhibited by methionine.