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Optimization of the cultivation media for thermophilic bacterium Caldimonas thermodepolymerans
Fajtová, Zuzana ; Černayová, Diana (referee) ; Obruča, Stanislav (advisor)
The subject of the bachelor ‘s thesis is the optimization of the cultivation conditions of the thermophilic bacterium Caldimonas thermodepolymerans. The theoretical part provides a general characterisation of PHA materials, their synthesis, degradation, and applications. In addition, extremophilic microorganisms and the bacterium C. thermodepolymerans are discussed in the theoretical part. The experimental part of the thesis focuses on the production of the PHB by the bacterium C. thermodepolymerans DSM 15344. A series of experiments were carried out to optimise the cultivation conditions. The influence of temperature, inoculum ratio, inoculum age, mineral medium composition, and concentrations of nitrogen sources in the mineral medium on culture growth and PHB production was investigated. It was found that the original conditions set were already close to optimal for the cultivation. A higher yield compared to the control conditions was observed with a combination of 0.5 g/l yeast extract and 1.5 g/l ammonium chloride, resulting in a 7% increase in PHB.
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Use of molecular techniques for study of thermophilles
Dvořáková, Dominika ; Kouřilová, Xenie (referee) ; Buchtíková, Iva (advisor)
Polyhydroxyalkanoates are microbial storage polymers that represent a green alternative to petrochemical plastics. However, their high production cost limits wider industrial utilization. Use of waste materials as a substrate and/or utilization of extremophilic organisms is an option to reduce production costs. The strain of thermophilic bacteria Aneurinibacillus appears to be a promising producer because of its ability to synthesize wide range of non traditional copolymers. In addition, PHA synthesis is independent of nutrient limitation, which was the subject of this study. Experiments were focused on the expression of genes involved in PHA synthesis in the bacterium Aneurinibacillus sp. AFn2 on different types of production media. The presence of selected genes was verified using classical PCR. After that the cultivation experiments were performed on mineral and complex media to simulate environments with different nutrient availability. Biomass concentration was determined gravimetrically from the samples collected in time during the growth curve measurement. PHA content was measured simultaneously using gas chromatography. Finally, the expression of the studied genes was analysed by RTqPCR. Significant differences were observed in the transcription of individual genes depending on the media type and time. The results confirmed the ability of Aneurinibacillussp.AFn2 to produce PHA independently on nutrient limitation, however, the production rate and cell filling depended on the media type. This fact may be related to the different course of expressed genes involved in PHA synthesis. A higher percentual yield of P(3HB) in cells were observed in complex media along with an increasing trend in the overall gene expression. On the other hand, in mineral medium, the transcription of individual genes only decreased after initial strong expression, and the cell filling of P(3HB) was also lower. The findings lead to understanding not only the expression of selected genes but also the overall synthesis of PHA. At the same time, it offers the way to optimize production with the aim of reducing cost and expanding their market applications.
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Study of the production of polyhydroxyalkanoates using selected thermophilic representatives of the genus Aneurinibacillus
Řeháková, Veronika ; Kovalčík, Adriána (referee) ; Pernicová, Iva (advisor)
The subject of the diploma thesis is the production of PHA copolymers by thermophilic Gram-positive bacteria of the genus Aneurinibacillus. The theoretical part is devoted to the general characterization of PHA materials, their properties, use, and also to the production of PHA polymers by these bacteria. The experimental part deals with the production of PHA copolymers using selected thermophilic members of the genus Aneurinibacillus. Firstly, the specific enzyme activity of PHA synthases was determined, and then the production of PHA copolymers was screened using selected lactones (-valerolactone, -hexalactone, -valerolactone) and diols (1,6-hexanediol, 2,3- butanediol and 1,4-butanediol). These experiments were performed with six thermophilic producers by the members of the genus Aneurinibacillus, which were isolated from compost and activated sludge. Tested microbial strains have demonstrated the ability to integrate interesting monomers into the PHA structure, including 4-hydroxyvalerate (represented up to 69.3% of the total PHA), 5-hydroxyvalerate (up to 47.1%), or 4-hydroxyhexanoate (up to 31.9%). Subsequently, a closer characterization of the obtained PHA (which were gained by the production of the best producers) using advanced methods (DSC, SEC-MALS, FT-IR) was performed. Finally, the screening of the PHA copolymers production was performed in laboratory bioreactors.
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Thermophilic microorganisms application to biodegradation
Varmužová, Tamara ; Márová, Ivana (referee) ; Babák, Libor (advisor)
This thesis is focused on study of biodegradability of modified polyurethane elastomeric films in synthetic medium with minerals and vitamins on tempered shaker by mixed thermophilic aerobic bacterial culture Bacillus and Thermus genera. In most cases addition of all used fillers (carboxymethyl cellulose, hydroxyethyl cellulose, acetylated cellulose, acetylated starch and glutein) led to increased biodegradability of elastomeric films with modifying agent in comparison with elastomeric films without modifying agent (referential). The growth of cultures was strongly increased in presence of elastomeric films modified by 10 % acetylated cellulose and 10 % carboxymethyl cellulose. Elastomeric film biodegradation mechanism included probably two stages: abiotic destruction of elastomeric films and consequent utilization of degradation products by bacterial culture.
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Identification and isolation of PHA producing bacteria
Pernicová, Iva ; Ondrejovič, Miroslav (referee) ; Rychtera, Mojmír (referee) ; Obruča, Stanislav (advisor)
Polyhydroxyalkanoates (PHA) are microbial storage polyesters that represent a renewable and environmentally friendly alternative to petrochemical plastics. However, their production and use are severely disadvantaged by the high production cost. The use of extremophilic PHA producers is one of the ways to reduce the cost of PHA production. Extremophiles bring numerous advantages resulting from the high robustness of the process against microbial contamination. In this doctoral thesis, attention was focused on the study of PHA production using selected halophilic and thermophilic microorganisms. Representatives of the genus Halomonas were mainly from public collections of microorganisms. Two promising PHA producers on waste frying oil were identified, namely Halomonas hydrothermalis and Halomonas neptunia. Both strains achieved good PHA yields in flask experiments. With the addition of suitable structural precursors, they were also able to produce copolymers with interesting material properties. However, in the proposed thesis, the main emphasis was placed on the study of PHA production using thermophilic microorganisms. As a part of the work, the isolation of thermophilic PHA producers from various thermophilic consortia (active sludge, compost, etc.) was performed. During isolations experiments, an original isolation procedure was designed using changes in osmotic pressure, the so-called osmoselection. Dozens of promising thermophilic PHA producers were obtained thanks to this original approach. They were taxonomically classified using 16S rRNA and tested for production potential. The most promising PHA producer was the isolate which was classified as Aneurinibacillus sp. H1. This bacterium is able to utilize a variety of substrates, including waste glycerol, to produce PHA. Even more important is the capability of synthesizing copolymers with a high content of 4-hydroxybutyrate. The monomer composition of the PHA copolymer and thus the material properties of the prepared copolymer can be controlled by suitable adjustment of the cultivation conditions. The prepared copolymer P(3HB-co-4HB) has unique properties and the great application potential in numerous high-end applications, for example in the field of health care, food industry or cosmetics.
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Molecular characterization of selected PHA producers
Kubáčková, Eliška ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
This diploma thesis focuses on the molecular characterization of selected PHA producers. Within this work, the PHA producing thermophilic isolates originating from the samples of activated sludge and compost were identified and characterized using molecular biological methods. By sequencing the 16S rRNA gene, the thermophilic isolates were identified and taxonomically classified into the Firmicutes bacterial phylum. In these bacterial isolates, the ability to produce PHA at the genotype level was determined by conventional PCR detection of the phaC gene encoding PHA synthase, which is a key enzyme in PHA biosynthesis. Class I, II and IV PHA synthases were detected in most of the isolated bacteria, wherein class I and II PHA synthases are not characteristic for these bacterial genera. The largest proportion of isolates was identified for the species of thermophilic bacterium Aneurinibacillus thermoaerophilus, in which class IV PHA synthase was detected. In the second part of the diploma thesis, the RT-qPCR method was implemented to study the expression of selected genes of the bacterium Cupriavidus necator H16 involved in PHA metabolism. As part of the implementation of this method, PCR-based detection of selected genes was optimized and quantification of genes using real-time PCR was performed. The tested method included steps of RNA isolation, cDNA synthesis and quantification of gene segments for which the critical points of the method were determined based on the obtained data.
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Screening of biosurfactant production in selected thermophilic bacteria
Řeháková, Veronika ; Nováčková, Ivana (referee) ; Obruča, Stanislav (advisor)
The subject of the bachelor thesis is the production of biosurfactants by selected thermophilic bacteria. The theoretical part contains a general characterization of thermophilic bacteria, describes the structures and classification of biosurfactants, characterizes their properties and uses. The final part of the theoretical part deals with the production of biosurfactants by thermophilic bacteria. The experimental part deals with the screening of biosurfactant production by selected thermophilic bacteria. Thermophilic bacteria were obtained from composts and activated sludges. Isolation is not a subject of the thesis. In total 39 bacterial isolates were cultivated and their supernatants were tested by screening methods – emulsification capacity assay and solubilization of crystalline anthracene. Based on the results of these methods, 14 most promising producers were selected. These bacterial strains were re-cultured to obtain a new set of supernatants. For more detailed screening of production by selected thermophilic bacteria, in addition to determining the activity of the emulsion and solubilization of crystalline anthracene, other methods were added: direct surface tension measurement – Du-Noüy-Ring, drop pouring method and oil spreading method. The results were evaluated and the measurements were repeated for a new set of supernatants to verify the reproducibility of the production. The results of all the tests were compared and then three most reliable thermophilic bacterial producers were determined.
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Application of thermophilic Culture for Organic Compounds Biodegradation
Piechová, Jana ; Burdychová, Radka (referee) ; Babák, Libor (advisor)
The aim of this work is focused on biodegradability study of modified polyurethane materials (foams and foils) in glukose medium and biodegradation of whey. The biodegradability was tested with using mixed thermophilic aerobic bacterial culture of the Bacillus and Thermus genera. The cultivations were practised in the fermentor and in Erlenmayer flasks in a heated shaker. Maximal reduction of COD and culture growth has been achieved by biodegradation of polyuretane in the fermentor. Main effect of degradation was abiotic destruction of PUR materials and consequent utilization of degradation products by bacterial culture. These tests indicated that the best modification agent for biodegradation was 10% carboxymethyl cellulose. The polyurethane foam was more decomposed then the polyurethane foil. Biodegradation of whey without proteins in the fermentor was detected decrease of CHSK and lactose concentration about 50 % in 20th hour. Optimal conditions for whey biodegradation with the thermophilic bacterial culture were pH 6,5 and temperature 60 °C.
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Employment of thermophilic bacteria for biodegradation of synthetic and natural polymers
Csölle, Eduard ; Sedlář, Karel (referee) ; Obruča, Stanislav (advisor)
This bachelor’s thesis deals with the employment of thermophilic bacterium Schlegelella thermodepolymerans for biodegradation of selected polymers, namely poly(3-hydroxybutyrate) (PHB), polylactic acid (PLA), and poly(butylene adipate-co-terephthalate) (PBAT). The theoretical part focuses on the properties and origin of these bioplastics, the characteristics of the studied bacteria, and the general course of the biodegradation process. The experimental part discusses the twenty-day cultivation of three tested strains in the presence of the mentioned polymers and the subsequent evaluation of the degradation rate of these materials. The greatest increase in biomass content was observed by spectrophotometry for strain DSM 15344 when cultured on PHB. With blank samples (without polymers), it was shown that for significant growth, the bacteria had to truly utilize the polymers present. Gravimetric analysis of biomass content confirmed that PHB was the most suitable substrate for cultivation. The highest mass losses were recorded for PHB and were around 30 % across all three strains. For strain LMG 21645, the mass has decreased by almost 33 %. The weight losses of PLA and PBAT were considerably lower. Nevertheless, the most significant molecular weight loss was observed by SEC-MALS for PLA, reaching approximately 80 % for strain DSM 15264. SEM analysis of the polymer surface confirmed that the biodegradation of PHB occurs by enzymatic surface erosion. In contrast, the PLA and PBAT samples did not show any signs of biodegradation.
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Study on metabolic properties of the bacterium Schlegelella thermodepolymerans with respect to biosynthesis of polyhydroxyalkanoates
Chvátalová, Viktorie ; Kouřilová, Xenie (referee) ; Pernicová, Iva (advisor)
This bachelor's thesis deals with the metabolic properties of the thermophilic bacterium Schlegelella thermodepolymerans, which is capable of producing polyhydroxyalkanoates (PHA). The aim of the study was to understand the metabolism and utilization of different carbohydrates (xylose, cellobiose, glucose and their combinations) depending on the cultivation time. In the experimental part, two bacterial strains (M15344 and DSM15344) were used. The increase in optical density was monitored during the measurements for the bacteria. Furthermore, the content of P(3HB) from the biomass was determined using GC-FID and the molecular weight of the polymer was determined using SEC- MALS analysis. By measuring residual sugar using HPLC-RI, the course of carbohydrate utilization was determined as a function of time. The results of both bacteria were compared and evaluated. DSM15344 showed better results in P(3HB) production than M15344, and cellobiose and xylose appeared to be the most suitable substrates for P(3HB) production.
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