|
|
Analytical methods for determination of polyhydroxyalcanoates content in bacterial cells
Černayová, Diana ; Enev, Vojtěch (referee) ; Sedláček, Petr (advisor)
This bachelor thesis deals with methods that are used for determination of polyhydroxyalkanoates (PHA) in cells of bacteria Cupriavidus necator H16. To the applied methods belong Fourier transform infrared spectroscopy, Raman spectoscopy, turbidimetry and thermal analysis (thermogravimetric analysis and differential scanning calorimetry). The results of each method were compared to amount of PHA determinated by gas chromatography. Fourier transform infrared spectroscopy and Raman spectroscopy revealed a linear relation between ratio of characteristic peaks and amount of PHA. FTIR enabled to determine not only the quantitative amount of PHA, but the quality of the sample (cristallinity, amorphous state) as well. Another method was turbidimetry, which has shown correlation between dispersion of UV-VIS light to amount of PHA in bacterial cells. Thermogravimetric analysis was the most effective technique for determination of PHA. The dependence of weight loss of PHA on its content (found by gas chromatography) was determined with the highest accuracy to gas chromatography. Tha last method- DSC was not alternative replacement for quantitative PHA determination. However, DSC indicated crystalline and amorphous state of the polymer.
|
|
|
Photoprotective properties of PHB granules
Hesko, Ondrej ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
The objective of this work is study and design of physical-chemical methods for verification of photoprotective properties PHB inclusions using bacteria Cupriavidus necator H16 and its mutated bacteria strain Cupriavidus necator PHB-4 which differs in production capability of polyhydroxyalkanoates. Basic information about of polymer inslusions and methods used for proving photoprotectivity are discussed in theoretical part. Methods such as nephelometry and continous fluorescence microphotolysis (CMF), which is used to characterize the mechanism of interaction of light with inclusions, were optimized. In experimental part properties of light scattering in bacteria were determined and characteristic curves were obtained. Further the photostability of fluorescence probe specific for nucleic acids were compared in bacteria strains. This method should prove bigger tolerance of bacteria H16 for UV light. It should also define possible interaction of nucleic acids with PHB inclusions.
|
|
|
Analytical centrifugation as a tool for characterization of microbial cells
Beránková, Barbora ; Müllerová, Lucie (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis deals with the study of centrifugation potential of microorganisms depending on the cultivation conditions The experimental part focuses on the sedimentation velocity and the instability index of the studied microorganisms during growth, under temperature and salt stress, the influence of the medium composition on centrifugation and the effect of osmotic strength on halophilic microbe Haloferax mediterranei. The sedimentation velocity and the instability index are increasing with increasing amount of water present in the bacterial culture of Haloferax mediterranei. The presence and type of polyhydroxyalkanoate (PHA) play a role in the monitoring of growth of bacteria Cupriavidus necator H16, Cupriavidus necator PHB-4, Haloferax mediterranei, Burkholderia cepacia and Burkholderia sacchari at the rate of sedimentation and instability index. Bacteria Cupriavis necator H16 with the highest content of poly(3-hydroxybutyrate) present was the fastest sedimentating and most unstable of the bacteria studied. Bacteria Cupriavidus necator H16 and Cupriavidus necator PHB-4, exposed to temperature and salt stress, exhibited only a slight influence on the rate of sedimentation and instability under temperature stress. The sedimentation velocity and instability index decreased with saline stress with increasing concentration of NaCl solution in culture. When comparing the media composition to polyhydroxyalkanoate (PHA) producing bacteria, Cupriavidus necator H16 cultivated in the mineral medium demonstrated the lowest sedimentation velocity and instability index rates, but Cupriavidus necator PHB-4 reached the lowest sedimentation velocity and instability index when grown in Nutrient Broth medium. For yeast Saccharomyces cerevisiae, the choice of medium had only a slight effect on sedimentation velocity and instability index.
|
|
|
Stress resistance of bacterial pure cultures and mixed cultures
Chorvátová, Michaela ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
The purpose of this bachelor thesis was to investigate a stress resistance of bacterial monocultures and defined mixed culture. The theoretical part contains a literary review, which deals with the general stress resistance of bacteria and the dynamics of their growth. Furthermore, polyhydroxyalkanoates are introduced, as well as their involvement into stress response of bacteria. In this review was also elaborated the flow cytometry, significant modern method, which was important part of the experiment. In the experimental part, the resistance to osmotic and temperature stress was studied. For this purpose, monocultures of the PHA accumulating strain Cupriavidus necator H16 and the mutant strain Cupriavidus necator H16/PHB-4 unable to accumulate PHA were used. The defined mixed culture was prepared by cocultivating these two strains. Overall, the accumulation and utilization of PHA, namely poly (3-hydroxybutyrate), increased the resistance of bacteria. The most notable was the increase in the case of osmotic stress, when salt concentrations did not significantly reduce the viability of Cupriavidus necator H16 against a mutant strain which viability decreased with growing intensity of stress. On the other hand, experimental temperatures did not have a significant effect on cell viability. The highest growth of bacteria was recorded, in most cases, at their temperature´s optimum of 30 °C.
|
|
|
Biotechnological production of selected biopolymers employing Haloferax mediterranei
Strečanská, Paulína ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
Aim of this bachelor thesis is to study of production of selected biopolymers, polyhydroxyalkanoates (PHA) and extracellular polysaccharides (EPS) by Haloferax mediterranei. The first part contains theoretical background including properties of biopolymers, advantages and purposes of commercial production, characteristics and positive aspects of cultivation of extreme halophile archaea Haloferax mediterranei. The experimental part is focused on study of effects of concentration of waste substrates, such as proteolytic whey hydrolysate and feather hydrolysate on PHA and EPS production. The effect of medium volume and its correlation with acceess of oxygen on biopolymer production were studied as well. PHA, in particul copolymer P(3HB-co/3HV) was analysed by gas chromatography with FID detection. The content of 3HV in PHA to 15,09 % was achieved using medium which contains 25 % proteolytic whey hydrolysate without glucose supplementation. Medium with 10 % of feather hydrolysate and 10 % of feather hydrolysate with 30 g/l glucose proved to be suitable in volume 50 ml in 250 ml Erlenmeyer flask. Haloferax mediterranei was able to produce biomass and accumulate PHA using protein substrate without need of saccharide supplementation.
|
|
|
Bioproduction, Characterization and Application of Selected Biopolymers
Benešová, Pavla ; Pekař, Miloslav (referee) ; Čertík, Milan (referee) ; Márová, Ivana (advisor)
Proposed doctoral thesis deals with microbial production of biopolymers (polysaccharides and polyesters) using renewableble inexpensive resources as carbon and nitrogen source. At first, production of extracellular metabolites, mainly pullulan and poly-L-malic acid, by polymorphic microorganism Aureobasisium pullulans was studied. Further part of thesis was focused on production of bacterial biopolyesters polyhydroxyalkanoates (PHA). PHA are produced in the form of intracellular granules by various bacteria species. During the study of PHA production, sythesis of poly(3-hydroxybutyrate) and technologically significant copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was investigated as well with regard to waste substrate utilization in biorafinery concept. Spent coffee ground seemed to be applicable waste in biorefinery proces. One of the coffee wastes used for PHA production was spent coffee ground hydrolysate which was converted to PHA by Burkholderia cepacia. Moreover, copolymer of 3HB and 3HV was accumulated when SCGH was used as a sole carbon source, no precursor had to be added. Another coffee waste, which was utillized by Cupriavidus necator H16, is represented by oil extracted from spent coffee grounds. Hydrolysis proces of waste substrates had to be optimized, to reach the highest possible saccharide concentration. Due to the hydrolysis process several toxic compounds can be formed, hence, influence of hydrolysis procesings on polyphenols content in hydrolysates was monitored. Waste substrates were also tested as potentional complex nitrogen source for PHA production. Hydrolysates of cheese whey and chicken feather were used as inexpensive complex nitrogen source for PHA production by C. necator, when waste frying oil was used as a carbon substrate. The finnal part of the thesis is dealing with possibilities how the PHA can be processed into atractive aplication forms. Liposomes with partial content of PHB had been prepared, moreover, for improving of antioxidant activity and antimicrobial effect the waste coffee oil was added. Micro-fibrous PHB materials were prepared for the purposes of water filtration. Higher filtration effectivity was observed when composite PHB fibrous material containing active charcoal or metal oxides was used. Important part of thesis is represented by study of degradation processes of prepared PHA-based aplication forms. Various microorganisms were tested on PHA depolymerases production. Biodegradability of PHA materials was tested in of composting conditions by standard composting test IS/ISO 20200. Changes in mechanical properties of polyurethan, where polyol was partialy replaced by PHB, were tested depending on the exposure of composting condition.
|
|
|
Polyhydroxyalkanoates and their role in bacterial biofilms
Rucká, Markéta ; Samek,, Ota (referee) ; Obruča, Stanislav (advisor)
This master thesis deals with polyhydroxyalkanoates (PHA) and their role in bacterial biofilms. In the theoretical part the polyhydroxyalkanoates, bacterial biofilm and the relationship between them were reviewed. The experimental part focused on differences in PHA production by planktonic and biofilm cells. In order to study selected topic, bacterial strains of Burkholderia cepacia and Burkholderia sacchari were cultivated using a CDC biofilm reactor. The attention was paid to quantity and especially to the form in which PHA occurs in planktonic and biofilm cells. Results of Raman spectroscopy have shown that PHA exists exclusively in native amorphous form in planktonic bacterial cells. On the other hand, in biofilm PHA occurs also in a partially crystalline form. In addition, the resistance of planktonic and biofilm cells against various stress factors and the effect of osmotic stress on PHA production was tested too. According to the results of the experiment, when the bacteria were exposed to different stress factors (high temperature, low temperature, presence of detergent and so forth) biofilm cells showed a higher stress resistance than planktonic cells. Apart from slowing cell growth and reproduction, increased osmotic pressure in the culture medium also caused decrease of PHA production. In addition, planktonic cells responded to external stimuli more sensitively than biofilm ones.
|
|
|
Study on protective effect of 3-hydroxybutyrate and its derivatives
Černá, Klára ; Smilek, Jiří (referee) ; Obruča, Stanislav (advisor)
The aim of this thesis is the study of chemical chaperones, as a preservative enzymes before denaturing processes. In the experimental part of the study we investigated the protective effects of five selected potential protective compounds – derivatives of 3-hydroxybutyrate – two model enzymes: lipase and lysozyme. Protective effects of potential chemical chaperones were investigated by dynamic light scattering (DLS), which was determined by the size distribution of the aggregates lipase produced depending on the temperature at the heat-induced denaturation. Further we have been used differential scanning calorimetry (DSC), which was determined by the temperature of the denaturation of lysozyme in the presence or absence of potentially protective substances. The last parameter was to determine the effect of potentially protective agents at different concentrations on the catalytic activity of lipase and determining the residual lipase activity after application of denaturing two factors – the high temperature and freezing. Of all tested structural analogues of 3-hydroxybutyrate was the highest protective effect observed (stabilization of enzyme molecules) with the succinate, which has two carboxyl groups. Conversely, 1,3-butanediol showed virtually no protective activity, indicating that the presence of carboxyl groups on the protective effect of essential. The low protective efficacy butyrate further indicates that it is essential that an effective protectant in addition to the carboxyl group contained as a further functional group – either carboxyl or hydroxyl. More significant protective effect was observed in 3-hydroxybutyrate than in 2-hydroxybutyrate. Interestingly, the effective protectant while at higher concentrations strongly inhibit the enzyme activity of the model enzyme, which is probably related to the solvation and conformations of the protein in the presence of protectant and the availability of the active site for the substrate.
|
|
|
Production of polyhydroxyalkanoates employing Haloferax mediterranei
Hlináková, Kristýna ; Kučera, Dan (referee) ; Obruča, Stanislav (advisor)
This bachelor thesis is focused on study of selected effects on production of both polyhydroxyalkanoates (PHA) and extracellular polysaccharides (EPS) by halophile Archae Haloferax mediterranei. Theoretical part deals with a brief characterization of biopolymers and provides overwiev of the relevant microbial producers. The experimental part is focused on selection of a suitable substrate, concentration of sodium chloride and glucose in culture medium for a optimal production of biopolymers. Effects of light and suitable cultivation temperature were studied as well. UV-VIS spectrophotometry and gravimetry were used for analysis of biomas content. The content of PHA was analysed by gas chromatography with FID detection. PHA produced by H. mediterranei is a copolymer P(3HB-co-3HV), 3HV content is about 10%. The mixture of glucose and glycerol proved to be the most suitable substrate for a production of PHA, with a content od 80,6% of PHA. Further, we investigated optimal concetration of NaCl, which was found at 200 g/l. Interestingly, microbial culture responded to various salt concertations by differences in pigment formation. Finally, the last parameter optimalized in this work was temperature of cultivation. The highest content of PHA was obtained at 37°C, but the culture was capable of PHA accumulation and growing also at 50°C.
|
|
|
Utilization of spent wood chips for biotechnological production of PHA
Ladický, Peter ; Sedláček, Petr (referee) ; Obruča, Stanislav (advisor)
The aim of this work was to study the suitability of wood shavings and sawdust as a substrate for microbial production of PHA by bacteria Burkholderia cepacia and Burkholderia sacchari. In the experimental part of the work the most appropriate approach of hydrolysis of wood shaving and sawdust and the effect of polyphenol and furfural concentration on ability to accumulate PHA was studied. Burkholderia sacchari had greater ability to accumulate PHA compared to Burkholderia cepacia. PHB values 87–89 % were achieved when Bulkholderia sacchari was cultivated on medium that contained detoxified hydrolysate of sawdust. Sawdust is therefore a promising substrate for microbial production of PHA in terms of reducing production costs and high content of PHB in biomass.
|
guest ::
