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Bioremediation of persistent aromatic pollutants
Stella, Tatiana ; Cajthaml, Tomáš (advisor) ; Mihaljevič, Martin (referee) ; Tesařová, Eva (referee)
The remediation of persistent chlorinated aromatic compounds has become a priority of great relevance due to the teratogenic, carcinogenic and endocrine-disrupting properties of these xenobiotics. The use of biological methodologies for the clean-up of contaminated sites, collectively referred to as "bioremediation", has been gaining an increasing interest in recent years because it represents an effective, cost-competitive and environmentally friendly alternative to the physico-chemical and thermal treatments. In this respect, "white rot" fungi, an ecological subgroup of filamentous fungi, display features that make them excellent candidates to design an effective remediation technology ("mycoremediation"). In spite of this, fungi have not been widely exploited for their metabolic capabilities and the mechanism by which they are able to degrade the aforementioned pollutants has not been fully elucidated yet. Within this frame, the present Ph.D thesis was aimed at: i) assessing the efficiency of different mycoremediation strategies for the clean-up of a polychlorinated biphenyl (PCBs)-contaminated soil; ii) understanding the fungal degradation pathways of polychlorinated biphenyls and their major metabolites, namely chlorobenzoic acids (CBAs) and hydroxylated polychlorinated biphenyls (OH-PCBs). i)...
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Production and characteritzation of extracellular hydrolases from selected moulds
Skoumalová, Petra ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
This diploma thesis is focused on study of potential production of extracellular hydrolytic enzymes. The theoretical part deals with characterization of selected hydrolytic enzymes, their catalytic properties, the possibility of extracellular hydrolase production by fungi and their applications. In experimental part production strains Aureobasidium pullulans, Fusarium solani and Phanerochaete chrysosporium were used. Productions of cellulase, amylase, xylanase, lipase, protease and lignin-degraded enzymes (laccase, manganese- dependent peroxidase, lignin peroxidase) were observed. Cultivations were carried out in submersed mode in mineral medium supplemented by waste co-substrates such as wheat bran, corn bran, rice bran and oat bran, sawdust, rice, apple fiber, egg pasta and egg-free pasta. Production of enzymes depended on the substrate type and time of cultivation. The highest cellulase, xylanase and amylase activities were measured in the first period of cultivation (3 to 7 day). Lignin-degraded enzymes and proteases were produced at the end of cultivation (7 to 10 days). Lipolytic activity was detected only in A. pullulans, where the activity increased with time of cultivation. The highest value was determined during cultivation on wheat bran (3.6 nmol/ml.min). The highest xylanase and celulase activity (170.3 nmol/ml.min, 248.0 nmol/ml.min) were determined during cultivation of F. solani on corn bran. The highest amylase activity (111.8 nmol/ml.min) was reported in P. chrysosporium during the cultivation on rice. The highest protease activity (68.0 nmol/ml.min) was determined in F. solani grown on wheat bran. The best producer of laccase was A. pullulans, the highest production was recorded for egg-free pasta (27.0 nmol/ml.min). The maximum lignin peroxidase activity (12.5 nmol/ml.min) was measured during the cultivation of F. solani on egg pasta, while the highest yield of Mn-dependent peroxidase (7.7 nmol/ml.min) was achieved during the cultivation of A. pullulans on wheat bran. Lignin-degraded enzymes behaved as inductive, while the other enzymes were produced in mineral medium too. Activity of cellulase in the mineral medium was in A. pullulans strain higher than in media with waste substrates. Enzymes produced into A. pullulans medium were purified by ultrafiltration, ion exchange chromatography and gel filtration.
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Biodegradace azobarviv působením hub bílé hniloby
Bírošíková, Paulína
White rot fungi, belonging to wood decay fungi have the ability to degra-de complex aromatic structures such as lignin or pollutants with an aromatic structure (tetracyclines, endocrine disruptors) via extracellular ligninolytic enzymes. Azo dyes are synthetic dyes containing aromatic rings with bonded substituents lin-ked by an azo group. Fungi are able to degrade these structures, whereas decrease of dye is observed, called decolorization. The aim of this bachelor's thesis is to de-termine the degradation ability of three types of azo dyes (methylene red, tartrazine and azorubin) using ten types of white rot fungi. Cultivation of individual fungi with the dyes was carried out for twenty-four days, during which the absorbance was monitored using a UV/VIS spectrophotometer at intervals of every other working day. The colour loss was calculated from the measured values and the decoloriza-tion values were expressed graphically as a percentages. The selected fungi showed the greatest decolorization of tartrazine on the second day of the experiment, an average of 89%, except for the fungus Fomes fomentarius, for which dye decoloriza-tion was not observed. In contrast, the least degraded dye was azorubin, an average of 37% at the end of the experiment for most fungi species. Methylene red showed the most stable decolorization trend, with an average of 77% at the end of the me-asurement. The effect of decolorization was influenced by the structure of the azo dyes and the efficiency of specific fungal species.
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Study on biodegradation of PCBs by white-rot fungi.
Kožená, Lenka ; Bosáková, Zuzana (advisor) ; Olšovská, Jana (referee)
The biodegradation of Delor 103, a commercial mixture of PCB congeners, was studied with eight strains of white rot fungi in two nutritive media: in mineral nitrogen limited medium (so called Kirk medium) and in complex Malt- extract glucose medium. The most efficient biodegradation was observed with the fungus Pleurotus ostreatus, where 99% of PCB congener sum was degraded after 42 days in both media. Bjerkandera adusta was able to degrade 89% of PCB congener sum within 42 days in complex medium. Irpex lacteus removed 70% of PCB congener sum within 42 days in Kirk medium. Other white rot fungi showed lower biodegradation ability, degrading only low chlorinated biphenyls. Further, urgent toxicity of samples was monitored during the Delor 103 biodegradation. P. ostreatus proved to be the most suitable agens for PCB decontamination as a significant reduction of toxicity in comparison with controls was observed during the PCB degradation. In case of other fungi, no reduction of urgent toxicity was observed showing a disadvantage of their use for PCB biodegradation. Activities of ligninolitic enzymes during biodegradation were also studied. In both media laccase activity was detected in Dichomitus squalens, Pycnoporus cinnabarinus and Trametes versicolor cultures. Ligninperoxidase enzyme activity was...
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Activities of enzymes involved in transformation of polycyclic aromatic hydrocarbons during composting
Šírová, Kateřina ; Cajthaml, Tomáš (advisor) ; Dračínská, Helena (referee)
Polycyclic aromatic hydrocarbons (PAHs) are recalcitrant organic pollutants, which occur widely in the environment. Some of these compounds are carcinogenic and toxic, many studies therefore focus on suitable remediation technologies. It has been shown that composting is an efficient treatment for contaminated solid matrices. Changes in several enzyme activities during co-composting of PAH-contaminated soil were studied in this thesis. The total initial concentration of analyzed PAHs in the soil was 1065 ± 86 µg·g-1 . The chosen activities represented well-known key enzymes involved in the transformation of PAHs or catechol as the central metabolite of PAH microbial degradation. At first, a method for extraction of the selected enzymes from the compost matrix was optimized. This approach was then used for the extraction of the enzymes from compost samples collected at each phase of composting. The activity of manganese peroxidase, laccase, tyrosinase and catechol-2,3-dioxygenase was detected during the cooling and the maturation phase. The only detected activity during the initial mesophilic phase was that of manganese peroxidase. The activities of catechol-1,2- dioxygenase and lignin peroxidase were not detected at all. Despite the fact that PAHs were substantially degraded, no influence of PAHs...
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Imobilizácia lignolytických enzýmov
Schlosserová, Nikola
Ligninolytic systems found their application in the food industry, but they are also used for the degradation of several xenobiotic compounds and dyes. This characteristic is making them be a useful tools for bioremediation purposes, that is why the interest in ligninolytic enzymes is increasing. The main aim of practical part was preparation of immobilized enzyme lignin peroxidase by method CLEA from fungus Piptoporus betulinus, Trametes gibbosa and their cocultivation. Activities of free and immobilized enzymes from these fungus were compared and measured by UV/VIS spectrometer. Conditions of precipitation and immobilization were specified by method CLEA. pH optimum for proteins precipitation were for Piptoporus betulinus pH 7,0, for Trametes gibbosa pH 5,0 and for cocultivation pH 6,0. The best concentration of glutaraldehyd, as a crosslinking agent, was for Piptoporus betulinus and cocultivation 50mM and for Trametes gibbosa 10mM. By immobilization, the activity of all enzymes were successfully increased up by 20 %. After optimalization of CLEA enzymes preparation, other parameters such as pH optimum, stability and temperature optimum and stability were tested. All enzymes had their pH optimum in acidic to slightly acidic environment and temperature optimum was in range from 30 °C to 40 °C. Immobilized CLEA enzyme from cocultivation was the most stable for all the enzymes. Free and CLEA enzymes were tested on synthetic food dyes, while their biodegradable ability was examined. Decrease of dyes was measured on HPLC with DAD detector. The sorption of fungi play an important role in this experiment, because fungi created mycelium and partially sorbed the dye. From this point of view, higher decolorization of dye in free enzymes in comparison with CLEA was observed. The best ability of decolorization was shown by a free enzyme from a Trametes gibossa, which after 14days of cultivation decolored more than 90 % dye. In total, significant decolorization (more than 80 % during 14 days) was achieved for azorubin. Degradation of dyes by CLEA was not so effective, which could be possibly caused by cultivation of enzymes without their substrate (veratrylalcohol). Cocultivation technique is very promising method how to increase LiP activity and concurrently create enzyme with improved properties.
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Využití hub bílé hniloby při bioremediaci polutantů
Nedvědická, Žaneta
Pharmaceutically active substances are a significant group of pollutants. Due to their presence in the environment and the difficult degradation, the interest of the scientific community in this issue has increased. An alternative to the current degradation processes of these substances is the action of white rot fungi, due to their effective non-specific enzymatic system. The practical part of my thesis focuses on the possibility of biodegradation with six white rot fungi strains Phellinus rhamni, Schizophyllum communae, Phellinus robustus, Ganoderma resinaceum, Phanerochaete chrysosporium and Trametes versicolor. The Trametes versicolor strain was selected based on a screening focused on the growth potential and degradation abilities. This strain was used in the experimental part of my thesis for the biodegradation of selected pharmaceutically active substances - namely in the form of the most widely used non-steroidal anti-inflammatory drugs sold over-the-counter: Aspirin and Paralen. During the experiments, the decrease of drug concentrations and the activity of lacasse as a representative of the ligninolytic enzymes involved in the degradation of pharmaceutically active substances were monitored spectrophotometrically over time. This was conducted in a nutrient medium under cultivation with Trametes versicolor. During experiments conducted 15 days, the fungus demonstrated the ability to break down both studied drugs, which was successfully proved by the 100% degradation in the medium. The decrease in drug concentrations was accompanied by an increase in laccase activity. The results showed that under the experimental conditions, laccase was an active extracellular enzyme. Biosorption and possibly intracellular enzymes also contributed to the removal of pharmaceutically active substances. Based on the measured data, Trametes versicolor is one of the best candidates for decontamination in practice and could be used to reduce the ecotoxicological impact of pollutants present in wastewater, food, textile, cosmetic and pharmaceutical industries.
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Using of modern molecular methods for isolation and identification of ligninolytic enzymes
Řiháček, Martin
Ligninolytic enzymes are able to decay the structure of lignin. This effect can be useful in the industry (food industry, textile industry, farming, etc) because it can replace regular chemical processes. The white-rot fungus Phanerochaete chrysosporium is known for the production of these enzymes. This bachelor thesis deals with the identification and characterization of the behavior of enzymes such as lignin peroxidase, laccase and manganese peroxidase under different concentrations of copper sulfate (0, 0.1, 0.5 and 1 mM). For isolation of DNA and RNA, the fungi were grown in potato dextrose broth (PDB) during 5 days. Common PCR, reverse transcription PCR and quantitative real-time PCR were chosen for this experimental part. The PCR products were purified and sent to sequencing to confirm how their different isoforms develop under stress conditions of different concentrations of copper sulfate. Moreover, the enzymatic activity assay for the enzymes was done also under different copper sulfate environment for the experimental part. 1 mM of copper sulfate concentration influenced the transcription of the enzymatic genes resulting in the production of their isoforms. It was also observed at the level of the gene expression, with a higher expression of these 3 genes; laccase, manganese peroxidase and lignin peroxidase, compared with the control samples. On the other hand, the best conditions for carrying out their enzymatic activities were observed at 0.5 mM concentration of CuSO4 for lignin peroxidase and manganese peroxidase and 1 mM in the case of laccase. After the molecular characterization, we can conclude that production of the enzymes of Phanerochaete chrysosporium are affected by high copper concentrations.
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Využití ligninolytických enzymů v potravinářství
Nadtochaeva, Polina
Ligninolytic enzymes are produced by wood-decaying fungi, and due to their high reactivity and low specificity, they have recently become important enzymes for biotechnological use. This thesis briefly reviews the most important ligninolytic enzymes and their reaction mechanisms and summarizes the ways of using these enzymes in the food industry. Great attention is paid to biosensors, which are used as modern analytical tools for the detection of phenolic compounds. In the experimental part, the own biosensor for the detection and determination of (+) catechin concentration was prepared. The measurement was carried out using a differential pulse voltammetry method using a carbon paste electrode prepared using the immobilized laccase from Trametes versicolor. Measurements of standard solutions have been performed to show that the biosensor can detect even relatively small amounts of catechin. Then the prepared biosensor was used for real samples of red wine and green teas. The content of catechin in four tea samples was measured. The concentrations were approximately the same and ranged from 19.57 to 24.91 mg·l-1. In the case of wine, it has been found that due to the complicated matrix, quantitative analysis is allowed, but requires appropriate mathematical processing of the results or pre-treatment of the samples.
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Degradace syntetických barviv pomocí Phanerochaete chrysosporium
Blahutová, Andrea
Azo dyes are the most important and largest group of commercially produced synthetic dyes. Their degradation by white-rot fungi Phanerochaete chrysosporium is induced by a lignin-degrading enzyme system, which is capable of degrading xenobiotic compounds. This bachelor thesis focuses on the degradation of the five most commonly used azo dyes by Phanerochaete chrysosporium. The process has been monitored not only on individual dyes (E102 Tartrazine, E110 Yellow, E122 Azorubin, E124 Ponceau 4R and E123 Amaranth), but also on two alimentary products (Vitacit – lemon and Vitacit – strawberry) containing E102 Tartrazine or E122 Azorubin azo dye. The absorbance of the solutions was measured spectrophotometrically at four-day intervals across a twenty-day cultivation period. The color loss was calculated based on the recorded values and the results were expressed as a percentage of decolorization. At the end of the measurement, the Azorubin dye showed the highest decolorization (90 %), whether the Ponceau 4R dye was decolorized the least (20 %). The level of decolorization of the powdered beverage solutions was compared with the decolorization of the individual dyes. The degradation was mainly effected by the various azo dyes structure. The composition of powdered beverages also played a role in their degradation.
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