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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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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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