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Bioinformatic analysis of PHA synthases of thermophilic bacteria
Brondová, Zuzana ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
The thesis deals with bioinformatics analysis, the aim of which was to find a suitable producer of PHA for new generation industrial biotechnologies from the collection of found thermophilic bacteria. Part of experiments was the finding of several thermophilic bacteria based on the similarity of the protein sequence of the phaC gene of the bacterium Cupriavidus necator. The next part of thesis was a literature search of the abilities of these thermophilic bacteria focused on culture conditions and the spectrum of usable substrates. Subsequently, five bacteria were selected for use in NGBI based on the information obtained. Freely available databases were used during the experimental work, and evolutionary analysis were performed in MEGA X and Operon-mapper. Rubrobacter xylanophilus with collection number DSM 9941 was selected from the collection of bacterial strains as the most promising PHA producer for NGIB. The high culture temperature of up to 70 ° C and a large amount of utilized carbohydrate substrates were considered decisive. An interesting result of the analysis was to find the gene sequences of two classes of PHA synthase – I. and III. class, as for a single bacterial strain from the entire collection. Additional genes linked to PHA metabolism were found in genome analysis.
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Employment of bacterium Tepidimonas taiwanensis for production of polyhydroxyalkanoates on waste substrates
Vidláková, Michaela ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
This diploma thesis is focused on screening PHA production using thermophilic bacterium Tepidimonas taiwanensis and on the study of possible use of grape pomace, molasses and waste paper as a cheap carbon substrate for culturing the characterized bacteria. At first, testing of basic cultivation parameters was performed, including carbon substrate concentration, oxygen availability, ability to utilize nitrogen sources and selected disaccharides. PHA production from waste substrates was tested in three ways. In the first, pre-prepared solids-free hydrolysates from raw materials were used as the carbon source. The second and third procedures were performed by dosing waste materials directly into the mineral media, which differed only in the presence or absence of the enzyme preparation enabling release of fermentable sugars. The most intensive increase in culture and the highest production of PHA was recorded on grape hydrolyzate. The biomass concentration in this sample reached up to 4.8 g/L with a content of 59 % PHA. On the other hand, the addition of grape marc directly to the production medium did not work at all, which was probably due to the presence of a large amount of inhibitory substances from the pomace. The situation was similar with molasses and waste paper, where the bacterium Tepidimonas taiwanensis was able to grow and possibly produce PHA only to a small extent. The work also managed to characterize the effect of temperature and pH on the activity of the enzyme cocktail Viscozyme L and to determine the temperature and pH optimum PHA synthase of the bacterial strain Tepidimonas taiwanensis in the cell lysate.
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Identification of PHA producing bacteria employing molecular techniques
Gajdová, Barbora ; Obručová,, Hana (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with identification of bacteria which are capable of producing polyhydroxyalkanoates (PHAs). Work included testing variety of genera including Pseudomonas, Lactobacillus, Bifidobacterium, thermophilic cultures and samples gathered from natural sources. Bacteria were investigated by molecular technique polymerase chain reaction – PCR. An amplification of the PHA synthase gene (phaC) was analyzed. In the first reaction phaC and 16S rRNA genes were tested at the same time. 16S rRNA gene is used as control for bacterial DNA and as an identification tool for natural source samples. This multiplex PCR used multiple primers in PCR mix. Second reaction search for amplicon specific for catalysing biosynthesis mcl-PHA (phaC1). The presence of the PHA synthase gene was verified in 11 samples which were Bifidobacterium breve CCM 7825T, Lactobacillus rhamnosus CCM 1825T, Lactobacillus zeae CCM 7069T, Lactobacillus delbrueckii subsp. bulgaricus CCM 7190T, Lactobacillus plantarum CCM 7039T, Pseudomonas gessardii, Pseudomonas fulva, Arthrobacter protophormiae, Curtobacterium flaccumfaciens, Mycobacterium neoaurum and Staphylococcus lentus.
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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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Isolation of PHA producing bacteria from mixed microbial consortia
Plachý, Petr ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
Aim of this bachelor thesis is detection of polyhydroxyalcanoates (PHA) producing bacteria from activated sludge and effort for isolation of these bacteria. The theoretical part deals with general issues of PHA and of bacterial production of PHA. Also there is attention paid to characterization of activated sludge and to selected methods used in this thesis for detection of PHA producing bacteria. In the experimental part, mixed microbial culture of the activated sludge was cultivated on different carbon sources. Potential PHA producers was isolated from these cultures with the use of lipophilic staining with Nile red and phaC gene (essential for PHA synthesis) was detected with the use of polymerase chain reaction (PCR) in 11 of the isolated cultures. By DNA sequencing 8 bacterial cultures were identified. It was Klebsiella pneumoniae (1 x), Paenirhodobacter enshiensis (1 x) and Pseudomonas putida (6 x). Presence of PHA in biomass was detected in 2 of the 11 isolated cultures by Fourier transformation infrared spectroscopy (FTIR) analysis. The content of polyhydroxybutyrate (PHB) was determined with the use of gas chromatography to 9,33 % of dry biomass (Paenirhodobacter enshiensis) and to 1,18 % (unidentified culture).
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Employment of bacterium Tepidimonas taiwanensis for production of polyhydroxyalkanoates on waste substrates
Vidláková, Michaela ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
This diploma thesis is focused on screening PHA production using thermophilic bacterium Tepidimonas taiwanensis and on the study of possible use of grape pomace, molasses and waste paper as a cheap carbon substrate for culturing the characterized bacteria. At first, testing of basic cultivation parameters was performed, including carbon substrate concentration, oxygen availability, ability to utilize nitrogen sources and selected disaccharides. PHA production from waste substrates was tested in three ways. In the first, pre-prepared solids-free hydrolysates from raw materials were used as the carbon source. The second and third procedures were performed by dosing waste materials directly into the mineral media, which differed only in the presence or absence of the enzyme preparation enabling release of fermentable sugars. The most intensive increase in culture and the highest production of PHA was recorded on grape hydrolyzate. The biomass concentration in this sample reached up to 4.8 g/L with a content of 59 % PHA. On the other hand, the addition of grape marc directly to the production medium did not work at all, which was probably due to the presence of a large amount of inhibitory substances from the pomace. The situation was similar with molasses and waste paper, where the bacterium Tepidimonas taiwanensis was able to grow and possibly produce PHA only to a small extent. The work also managed to characterize the effect of temperature and pH on the activity of the enzyme cocktail Viscozyme L and to determine the temperature and pH optimum PHA synthase of the bacterial strain Tepidimonas taiwanensis in the cell lysate.
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Bioinformatic analysis of PHA synthases of thermophilic bacteria
Brondová, Zuzana ; Brázda, Václav (referee) ; Obruča, Stanislav (advisor)
The thesis deals with bioinformatics analysis, the aim of which was to find a suitable producer of PHA for new generation industrial biotechnologies from the collection of found thermophilic bacteria. Part of experiments was the finding of several thermophilic bacteria based on the similarity of the protein sequence of the phaC gene of the bacterium Cupriavidus necator. The next part of thesis was a literature search of the abilities of these thermophilic bacteria focused on culture conditions and the spectrum of usable substrates. Subsequently, five bacteria were selected for use in NGBI based on the information obtained. Freely available databases were used during the experimental work, and evolutionary analysis were performed in MEGA X and Operon-mapper. Rubrobacter xylanophilus with collection number DSM 9941 was selected from the collection of bacterial strains as the most promising PHA producer for NGIB. The high culture temperature of up to 70 ° C and a large amount of utilized carbohydrate substrates were considered decisive. An interesting result of the analysis was to find the gene sequences of two classes of PHA synthase – I. and III. class, as for a single bacterial strain from the entire collection. Additional genes linked to PHA metabolism were found in genome analysis.
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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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|
|
Isolation of PHA producing bacteria from mixed microbial consortia
Plachý, Petr ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
Aim of this bachelor thesis is detection of polyhydroxyalcanoates (PHA) producing bacteria from activated sludge and effort for isolation of these bacteria. The theoretical part deals with general issues of PHA and of bacterial production of PHA. Also there is attention paid to characterization of activated sludge and to selected methods used in this thesis for detection of PHA producing bacteria. In the experimental part, mixed microbial culture of the activated sludge was cultivated on different carbon sources. Potential PHA producers was isolated from these cultures with the use of lipophilic staining with Nile red and phaC gene (essential for PHA synthesis) was detected with the use of polymerase chain reaction (PCR) in 11 of the isolated cultures. By DNA sequencing 8 bacterial cultures were identified. It was Klebsiella pneumoniae (1 x), Paenirhodobacter enshiensis (1 x) and Pseudomonas putida (6 x). Presence of PHA in biomass was detected in 2 of the 11 isolated cultures by Fourier transformation infrared spectroscopy (FTIR) analysis. The content of polyhydroxybutyrate (PHB) was determined with the use of gas chromatography to 9,33 % of dry biomass (Paenirhodobacter enshiensis) and to 1,18 % (unidentified culture).
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|
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Identification of PHA producing bacteria employing molecular techniques
Gajdová, Barbora ; Obručová,, Hana (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with identification of bacteria which are capable of producing polyhydroxyalkanoates (PHAs). Work included testing variety of genera including Pseudomonas, Lactobacillus, Bifidobacterium, thermophilic cultures and samples gathered from natural sources. Bacteria were investigated by molecular technique polymerase chain reaction – PCR. An amplification of the PHA synthase gene (phaC) was analyzed. In the first reaction phaC and 16S rRNA genes were tested at the same time. 16S rRNA gene is used as control for bacterial DNA and as an identification tool for natural source samples. This multiplex PCR used multiple primers in PCR mix. Second reaction search for amplicon specific for catalysing biosynthesis mcl-PHA (phaC1). The presence of the PHA synthase gene was verified in 11 samples which were Bifidobacterium breve CCM 7825T, Lactobacillus rhamnosus CCM 1825T, Lactobacillus zeae CCM 7069T, Lactobacillus delbrueckii subsp. bulgaricus CCM 7190T, Lactobacillus plantarum CCM 7039T, Pseudomonas gessardii, Pseudomonas fulva, Arthrobacter protophormiae, Curtobacterium flaccumfaciens, Mycobacterium neoaurum and Staphylococcus lentus.
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