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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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Methods for identification of PHA producing bacteria
Skřivanová, Veronika ; Turková, Kristýna (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with testing, optimazing and comparing methods for the identification of bacteria producing polyhydroxyalkanoates. Work included cultivation and microscopy methods, wherein the bacterial cells were stained with lipophilic dyes Nile red and Sudan black. Further, we also used flow cytometry and spectroscopic methods - Raman spectroscopy and infrared spectroscopy with Fourier transformation, and molecular biological methods, which analyzed the presence of a gene encoding PHA synthase (phaC) by polymerase chain reaction (PCR). PCR assay consist of two reactions, the firt on eis based on amplification of phaC gene along with 16S rRNDA gene, which is common for all the bacteria (multiplex PCR). The second reaction is focused on specific amplification of PHA synthase catalyzing biosynthesis of mcl-PHA. In order to overcome false positive results typical for methods analyzing genotype and also to avoid false negative results occuring in fenotype analyzing methods, the best strategy is to combine both aproaches. According to our results, analysis of presence of phaC gene by PCR can be combined with methods capable of determining presence of PHA in bacterial cells. For this purpose, Raman microspectroscopy seems to be very promising tool, since it is able to detect low content of PHA in cells and PHA can not be confused with other lipid metabolites. The results provide an overview of test methods, their advantages and disadvantages and also to compare different criteria according to which it is possible to choose the method of identification in depending on the adjustable requirements.
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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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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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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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Methods for identification of PHA producing bacteria
Skřivanová, Veronika ; Turková, Kristýna (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with testing, optimazing and comparing methods for the identification of bacteria producing polyhydroxyalkanoates. Work included cultivation and microscopy methods, wherein the bacterial cells were stained with lipophilic dyes Nile red and Sudan black. Further, we also used flow cytometry and spectroscopic methods - Raman spectroscopy and infrared spectroscopy with Fourier transformation, and molecular biological methods, which analyzed the presence of a gene encoding PHA synthase (phaC) by polymerase chain reaction (PCR). PCR assay consist of two reactions, the firt on eis based on amplification of phaC gene along with 16S rRNDA gene, which is common for all the bacteria (multiplex PCR). The second reaction is focused on specific amplification of PHA synthase catalyzing biosynthesis of mcl-PHA. In order to overcome false positive results typical for methods analyzing genotype and also to avoid false negative results occuring in fenotype analyzing methods, the best strategy is to combine both aproaches. According to our results, analysis of presence of phaC gene by PCR can be combined with methods capable of determining presence of PHA in bacterial cells. For this purpose, Raman microspectroscopy seems to be very promising tool, since it is able to detect low content of PHA in cells and PHA can not be confused with other lipid metabolites. The results provide an overview of test methods, their advantages and disadvantages and also to compare different criteria according to which it is possible to choose the method of identification in depending on the adjustable requirements.
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