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Study on metabolism of polyhydroxybutyrate and glycogen in cyanobacteria
Drinka, Jakub ; Slaninová, Eva (oponent) ; Obruča, Stanislav (vedoucí práce)
The submitted diploma thesis is focused on establishing a quantification method for glycogen analysis in cyanobacterial cells in order to be able to consider the impact of illumination and other parameters on accumulation of reserve polymers, glycogen and polyhydroxyalkanoates (PHA), namely poly(3-hydroxybutyrate) (PHB). The experiments were conducted with two cyanobacterial species, Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192, which were grown both in Erlenmayer flasks (EF) and multicultivator (MC). The methodology for glycogen accumulation was introduced based on available literature and conducted optimalization. The effect of different illumination conditions was observed in a nitrogen-limiting media M22O, in which half of the cultures were cultivated with a 16 hours of light and 8 lights of darkness periods (EF) for the whole duration of the experiment. Others were transfered into full-time dark period after entering the dormant chlorosis state, following the exhaustion of nitrogen levels in the media. Synechocystis sp. PCC 6803 showed a decrease in both of the reserve polymers accumulation when introduced to this type of stress conditions. On the other hand, Synechocystis salina CCALA 192 converted some of the glycogen into PHB in the dark, but the polyester levels were lower than those of the cultures continuously cultivated under the lamp. A negative effect on the biomass concentration was also detected, while cyanobacterial pigments seemed to be unaffected by the lack of light, their levels in the EF that remained illuminated decreased due to chrolosis. The experiments in the MC were conducted in the same way, but the light period consisted of constant, 24-hour illumination. Synechocystis sp. PCC 6803 seemed to follow a different trend than in cultivations in EF, the PHB concentration was not affected by the dark period and remained on the same amounts, while glycogen was metabolised. Synechocystis salina CCALA 192 increased its polyester reserves in the darkness and in comparison with the first species accumulated almost 4 times more PHB. However, the results acquired from cultivations in MC seemed to be very unequal due to a lot of small differences in the cultivation conditions. That was the reason why in the later stages of experiments they were focused more on a possible PHA copolymer formation, rather than comparing the functions of these two reserve polymers in the light/dark cycles. However, none of the cultivations was succesful in this matter and no monomer other than 3-hydroxybutyrate (3HB) was detected in the dried biomass.
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Study on PHa biosynthesis by cyanobacteria
Drinka, Jakub ; Kouřilová, Xenie (oponent) ; Obruča, Stanislav (vedoucí práce)
This bachelor thesis concerns with the effect of selected environmental stresses towards the viability of cyanobacterial species Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. Both strains were studied via flow cytometry using the appropriate fluorochromes while also observing the applicability of these stains and precision of this method in the analysis of cyanobacterial cultures. In spite of the optimization being carried out, fluorescein diacetate was not suitable for the analysis of these cells because of their autofluorescence interfere with the fluorochrome emissions. Therefore, SYTOXTM Blue Dead Cell stain was used for the further experiments. A cryoprotective effect of polyhydroxybutyrate was observed during the freezing-thawing cycles. On the other hand, the culture rich in PHB seemed to be less resistant towards the oxidative stress induced by the hydrogen peroxide, probably justifiable by the state of chlorosis. Furthermore, this thesis deals with the increase/decrease in biomass concentration and intracellular PHB pool as a response to the external carbon source addition, analysed by the gas chromatography paired with FID detection. The most intesive cell growth in Synechocystis sp. PCC 6803 was detected in the presence of sodium acetate combined with gamma-butyrolactone while the highest PHB content in the medium supplied with acetate and 1,4-butanediol. Nevertheless, Synechocystis salina CCALA 192 showed markedly higher polyhydroxybutyrate accumulation under both photoautotrophic and mixotrophic conditions, the only non-suitable substrate was gamma-butyrolactone.
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Study on metabolism of polyhydroxybutyrate and glycogen in cyanobacteria
Drinka, Jakub ; Slaninová, Eva (oponent) ; Obruča, Stanislav (vedoucí práce)
The submitted diploma thesis is focused on establishing a quantification method for glycogen analysis in cyanobacterial cells in order to be able to consider the impact of illumination and other parameters on accumulation of reserve polymers, glycogen and polyhydroxyalkanoates (PHA), namely poly(3-hydroxybutyrate) (PHB). The experiments were conducted with two cyanobacterial species, Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192, which were grown both in Erlenmayer flasks (EF) and multicultivator (MC). The methodology for glycogen accumulation was introduced based on available literature and conducted optimalization. The effect of different illumination conditions was observed in a nitrogen-limiting media M22O, in which half of the cultures were cultivated with a 16 hours of light and 8 lights of darkness periods (EF) for the whole duration of the experiment. Others were transfered into full-time dark period after entering the dormant chlorosis state, following the exhaustion of nitrogen levels in the media. Synechocystis sp. PCC 6803 showed a decrease in both of the reserve polymers accumulation when introduced to this type of stress conditions. On the other hand, Synechocystis salina CCALA 192 converted some of the glycogen into PHB in the dark, but the polyester levels were lower than those of the cultures continuously cultivated under the lamp. A negative effect on the biomass concentration was also detected, while cyanobacterial pigments seemed to be unaffected by the lack of light, their levels in the EF that remained illuminated decreased due to chrolosis. The experiments in the MC were conducted in the same way, but the light period consisted of constant, 24-hour illumination. Synechocystis sp. PCC 6803 seemed to follow a different trend than in cultivations in EF, the PHB concentration was not affected by the dark period and remained on the same amounts, while glycogen was metabolised. Synechocystis salina CCALA 192 increased its polyester reserves in the darkness and in comparison with the first species accumulated almost 4 times more PHB. However, the results acquired from cultivations in MC seemed to be very unequal due to a lot of small differences in the cultivation conditions. That was the reason why in the later stages of experiments they were focused more on a possible PHA copolymer formation, rather than comparing the functions of these two reserve polymers in the light/dark cycles. However, none of the cultivations was succesful in this matter and no monomer other than 3-hydroxybutyrate (3HB) was detected in the dried biomass.
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Study on PHa biosynthesis by cyanobacteria
Drinka, Jakub ; Kouřilová, Xenie (oponent) ; Obruča, Stanislav (vedoucí práce)
This bachelor thesis concerns with the effect of selected environmental stresses towards the viability of cyanobacterial species Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. Both strains were studied via flow cytometry using the appropriate fluorochromes while also observing the applicability of these stains and precision of this method in the analysis of cyanobacterial cultures. In spite of the optimization being carried out, fluorescein diacetate was not suitable for the analysis of these cells because of their autofluorescence interfere with the fluorochrome emissions. Therefore, SYTOXTM Blue Dead Cell stain was used for the further experiments. A cryoprotective effect of polyhydroxybutyrate was observed during the freezing-thawing cycles. On the other hand, the culture rich in PHB seemed to be less resistant towards the oxidative stress induced by the hydrogen peroxide, probably justifiable by the state of chlorosis. Furthermore, this thesis deals with the increase/decrease in biomass concentration and intracellular PHB pool as a response to the external carbon source addition, analysed by the gas chromatography paired with FID detection. The most intesive cell growth in Synechocystis sp. PCC 6803 was detected in the presence of sodium acetate combined with gamma-butyrolactone while the highest PHB content in the medium supplied with acetate and 1,4-butanediol. Nevertheless, Synechocystis salina CCALA 192 showed markedly higher polyhydroxybutyrate accumulation under both photoautotrophic and mixotrophic conditions, the only non-suitable substrate was gamma-butyrolactone.
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