Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

Orientador: Crispin Humberto Garcia Cruz / Banca: Alexandre Rodrigo Coelho / Banca: Vanildo Luiz Del Bianchi / Resumo: O alginato é um polissacarídeo normalmente extraído de paredes celulares de algas marrons utilizado nas indústrias de alimentos, farmacêuticas e biotecnológicas. A produção é concentrada no cultivo de algas marinhas marrons, mas várias bactérias do gênero Pseudomonas e Azotobacter produzem alginato. A estrutura química dos alginatos produzidos por algas é similar aos sintetizados pela A. vinelandii. Esta bactéria também produz polímeros intracelulares como o poli-hidroxibutirato (PHB), conhecido como bioplástico. Neste trabalho estudou-se a produção simultânea do alginato e PHB pela A. vinelandii utilizando sacarose, glicose e melaço de cana-de-açúcar como fontes de carbono, além de diferentes parâmetros de fermentação em agitador orbital rotatório. Os valores ótimos para a produção destes compostos foram determinados pela metodologia de superfície de resposta (MSR). O 1º experimento realizado para as três fontes de carbono foi um planejamento fatorial fracionado 26-2 (variáveis independentes: concentração da fonte de carbono; concentração de acetato de amônio; concentração de citrato de amônio e ferro (III); pH; temperatura de incubação e tempo de incubação). O 2º experimento baseou-se nos valores ótimos de produção de PHB para cada fonte de carbono e resultou em um planejamento fatorial completo 33-0 (variáveis independentes: concentração da fonte de carbono; temperatura de incubação e tempo de incubação). Verificou-se que a maior produtividade de PHB (100 mg/g de célula/h) utilizando o melaço de cana-de-açúcar ocorreu no tempo de incubação de aproximadamente 10 h, a 60,0ºC e nas concentrações de sólidos solúveis entre 14,0 - 25,0%. A glicose apresentou uma maior produtividade de PHB (60 mg/g de célula/h) no tempo de incubação de aproximadamente 10 h, entre 23,0-26,0ºC e concentração de glicose... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The alginate is a polysaccharide extracted from cell walls of brown seaweed used in the industries of food, pharmaceutical and biotechnology. The production is concentrated in the brown seaweed cultivation, but several bacteria, Pseudomonas and Azotobacter genus, produce alginate. The chemical structure of alginate produced by algae is similar to those synthesized by A. vinelandii. This bacterium also produces intracellular polymers such as polyhydroxybutyrate (PHB), known as bioplastic. In this work the simultaneous alginate and PHB production by A. vinelandii using sucrose, glucose and sugar cane molasses as carbon source, and different fermentation parameters in orbital shaker was studied. The optimum values for the production of these compounds were determined by the response surface methodology (RSM). The 1st experiment conducted for the three carbon sources was a fractionated factorial design 26-2 (independent variables: the carbon source concentration; ammonium acetate concentration; ammonium citrate and iron (III) concentration; pH; temperature and incubation time). The 2nd experiment was based on optimum values for the production of PHB for each carbon source and resulted in a full factorial design 33-0 (independent variables: the carbon source concentration; temperature and incubation time). The highest PHB yield (100 mg/g cell/h) using sugar cane molasses as a carbon source was found in 10 h at 60.0 ºC and solids soluble concentrations between 14.0 and 25.0%. The glucose showed the highest PHB yield (60 mg/g cell/h) in approximately 10 h, at temperature between 23.0 - 26.0 ºC and glucose concentration between 48.0 and 62.0 g/L. The sucrose, showed the highest PHB yield (45 mg/g cell/h) in approximately 18 h, 60.0 ºC and sucrose concentration of 10.0 g/L. For the alginate productivity using the glucose was observed that the yield was more... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:23:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-19Bitstream added on 2014-06-13T19:50:10Z : No. of bitstreams: 1 silva_an_me_sjrp.pdf: 5773853 bytes, checksum: 8d32b3256460ece4e4901a20da094ab8 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O alginato é um polissacarídeo normalmente extraído de paredes celulares de algas marrons utilizado nas indústrias de alimentos, farmacêuticas e biotecnológicas. A produção é concentrada no cultivo de algas marinhas marrons, mas várias bactérias do gênero Pseudomonas e Azotobacter produzem alginato. A estrutura química dos alginatos produzidos por algas é similar aos sintetizados pela A. vinelandii. Esta bactéria também produz polímeros intracelulares como o poli-hidroxibutirato (PHB), conhecido como bioplástico. Neste trabalho estudou-se a produção simultânea do alginato e PHB pela A. vinelandii utilizando sacarose, glicose e melaço de cana-de-açúcar como fontes de carbono, além de diferentes parâmetros de fermentação em agitador orbital rotatório. Os valores ótimos para a produção destes compostos foram determinados pela metodologia de superfície de resposta (MSR). O 1º experimento realizado para as três fontes de carbono foi um planejamento fatorial fracionado 26-2 (variáveis independentes: concentração da fonte de carbono; concentração de acetato de amônio; concentração de citrato de amônio e ferro (III); pH; temperatura de incubação e tempo de incubação). O 2º experimento baseou-se nos valores ótimos de produção de PHB para cada fonte de carbono e resultou em um planejamento fatorial completo 33-0 (variáveis independentes: concentração da fonte de carbono; temperatura de incubação e tempo de incubação). Verificou-se que a maior produtividade de PHB (100 mg/g de célula/h) utilizando o melaço de cana-de-açúcar ocorreu no tempo de incubação de aproximadamente 10 h, a 60,0ºC e nas concentrações de sólidos solúveis entre 14,0 - 25,0%. A glicose apresentou uma maior produtividade de PHB (60 mg/g de célula/h) no tempo de incubação de aproximadamente 10 h, entre 23,0-26,0ºC e concentração de glicose... / The alginate is a polysaccharide extracted from cell walls of brown seaweed used in the industries of food, pharmaceutical and biotechnology. The production is concentrated in the brown seaweed cultivation, but several bacteria, Pseudomonas and Azotobacter genus, produce alginate. The chemical structure of alginate produced by algae is similar to those synthesized by A. vinelandii. This bacterium also produces intracellular polymers such as polyhydroxybutyrate (PHB), known as bioplastic. In this work the simultaneous alginate and PHB production by A. vinelandii using sucrose, glucose and sugar cane molasses as carbon source, and different fermentation parameters in orbital shaker was studied. The optimum values for the production of these compounds were determined by the response surface methodology (RSM). The 1st experiment conducted for the three carbon sources was a fractionated factorial design 26-2 (independent variables: the carbon source concentration; ammonium acetate concentration; ammonium citrate and iron (III) concentration; pH; temperature and incubation time). The 2nd experiment was based on optimum values for the production of PHB for each carbon source and resulted in a full factorial design 33-0 (independent variables: the carbon source concentration; temperature and incubation time). The highest PHB yield (100 mg/g cell/h) using sugar cane molasses as a carbon source was found in 10 h at 60.0 ºC and solids soluble concentrations between 14.0 and 25.0%. The glucose showed the highest PHB yield (60 mg/g cell/h) in approximately 10 h, at temperature between 23.0 – 26.0 ºC and glucose concentration between 48.0 and 62.0 g/L. The sucrose, showed the highest PHB yield (45 mg/g cell/h) in approximately 18 h, 60.0 ºC and sucrose concentration of 10.0 g/L. For the alginate productivity using the glucose was observed that the yield was more... (Complete abstract click electronic access below)

To mitigate the problems of depleting and soaring price of fossil fuels, the production and use of renewable energy have been vigorously promoted. In Europe, the role of biologically-derived fuels and in particular biodiesel is gradually increasing in prominent. Rapeseed biodiesel is the most widely produced in Europe. As a consequence, enormous amount of by-products from production processes are being generated. Current strategies for managing these by-products (mainly rapeseed meal and crude glycerol) seem not to be economically sustainable. More efficient utilisation could add more value to the production chain which in turn would raise the competitiveness of biodiesel compared to petro-diesel. The aim of the project reported in this thesis was to study the feasibility of producing a value added product, polyhydroxybutyrate (PHB), from by-products generated from rapeseed biodiesel production processes as well as to investigate the effects of methanol, a major impurity in crude glycerol, on growth of Cupriavidus necator, a PHB-producing micro-organism.The preliminary study of C. necator growth in crude glycerol based media revealed that optimum concentration of crude glycerol was in a range 15-25 g/L. It was also found that slight changes in the carbon to nitrogen ratio of the feedstock did not significantly affect the growth while methanol at concentrations beyond 10 g/L did. A model based on a saturation equation was developed and used to successfully predict the inhibition of growth by methanol. From the developed model, mechanisms of the inhibition were proposed. The model could also be used to predict satisfactorily growth or productivity rates in other systems containing short-chain alcohols. The growth in solutions derived from rapeseed meal (designated as hydrolysate) via solid-state fermentation by Aspergillus oryzae followed by hydrolysis of the fermented solids was also studied. The biomass production was found to increase as a function of initial free amino nitrogen (FAN) concentration presented in the hydrolysate. However, at higher initial FAN concentrations, a lower conversion of nitrogen to biomass was observed. PHB production was studied using a feedstock which was a mixture of the hydrolysate and crude glycerol. Total biomass concentration reached 28.8 g/L at 120 h with 86% PHB content. PHB productivity and PHB yield on glycerol were 0.21 g/L•h and 0.32 g/g respectively. These results were comparable with those obtained when pure glycerol and synthetic crude glycerol were used, suggesting that, technically, the use of the generic rapeseed- and crude glycerol-based feedstock to produce PHB is feasible.Overall, the feasibility of producing PHB from rapeseed biodiesel by-products has been demonstrated. The satisfactory result leads to the more important outlook that the generic feedstock derived from rapeseed biodiesel by-products has the potential to be used to produce a wide range of products depending on the micro-organism used. Further development of this process to improve nutrient production efficiency as well as product yields and subsequent integration of the process into the biodiesel production process could well be an important contribution in the development of a sustainable biodiesel industry.

Polyhydroxyalkanoates (PHAs) are polyesters produced and degradable by prokaryotes, while biosurfactants/bioemulsifiers (BS/BE) are metabolites that reduces surface tension and synthesized by this group of organisms. Interest in the potential industrial applications for PHA and BS/BE has increased due to its eco-friendly appeal. In this study, 24 bacterial colonies were isolated from Atlantic forest soil and agro-industrial sludge (Coruripe-AL, Brazil) in minimum mineral medium, as indicative of the possible ability to synthesize PHA. All strains were submitted to biochemical characterization, while the phaC gene amplification showed that isolates BMA-05, BMA-10, BMA-13 and BDL-07 can express the key enzyme for the synthesis of PHA (PHA synthase). Sequencing of the partial 16S r-DNA region was able to identify these bacteria respectively as Pseudomonas fluorescens, Enterobacter aerogenes, Klebsiella oxytocaand Bacillus pumilus. The PHAs produced by each isolate were extracted with hot chloroform and the polymeric films were obtained. Experiments in shaken flasks (0 – 96 h) were conducted to compare the biomass, total reducing glycides, pH, and total protein in minimal medium containing different contents of glucose and peptone. Supplementation with peptone was able to induce the growth of these strains, and cell-free supernatant from the cultivation of Pseudomonas fluorescens BMA-05 showed no acidification in any of the conditions tested. To polyhydroxybutyrate production (evaluated after 24 h incubation), minimal medium was used with different nitrogen sources: ammonium acetate, ammonium chloride, ammonium nitrate, sodium nitrate, ammonium sulfate, beef extract, yeast extract, glycine and peptone. The maximum accumulation of P (3HB), detected by UV-visible spectrophotometry, was obtained for P. fluorescens, E. aerogenes, K. oxytoca and B. pumilus grown in ammonium sulfate, ammonium chloride, meat extract and nitrate sodium, respectively. Observations in transmission electron microscope showed Pseudomonas fluorescens BMA-05 with eletronlucent granules when the strain was cultivated in the presence of ammonium sulfate or sodium nitrate. Although no granule was observed for the other strains in the presence of ammonium sulfate, the gas chromatography analysis confirmed the production of P (3HB). When exoenzymes expression tests were conducted, the results indicated that the strains were able to hydrolyse gelatine, starch and Tween 80, and B. pumilus caused hemolysis of sheep blood. Experiments for biosurfactant/bioemulsifier production indicate that E. aerogenes BMA-10 excreted a compound surfactant which collapses the hydrophobic surface and emulsifies the hydrocarbons kerosene, toluene, diesel oil and soybean oil. The emulsification index (E24) using the cell-free supernatant of this microorganism was thermally stable in toluene, but not in kerosene. Similar results were obtained when the pH of this microorganism cell-free supernatant was adjusted to 2, 7 and 10, as well as different concentrations of NaCl (2, 6, and 10 %) were added to the samples. These results reveal the potential of Enterobacter aerogenes BMA-10 as a P(3HB)-producing, and its growth medium free of cells an emulsifier for biotechnological purposes and also in bioremediation. / Polihidroxialcanoatos (PHAs) são poliésteres produzidos e degradados por procariotos, enquanto biosurfactantes/bioemulsificantes (BS/BE) são metabólitos que diminuem a tensão superficial e sintetizados por esse grupo de organismos. O interesse nas potenciais aplicações industriais para PHA e BS/BE tem aumentado devido ao apelo ecologicamente correto desses produtos. No presente estudo, isolaram-se 24 colônias bacterianas de solo de Mata Atlântica e lodo agroindustrial (Coruripe-AL, Brasil) em meio mineral mínimo, como indicativo da possível capacidade de sintetizar PHA. Todas as linhagens foram caracterizadas bioquimicamente, enquanto a amplificação do gene phaC evidenciou que os isolados BMA-05, BMA-10, BMA-13 e BDL-07 podem expressar a enzima-chave para a síntese de PHA (PHA sintase). O sequenciamento da região parcial 16S r-DNA dessas bactérias permitiu suas identificações respectivamente como Pseudomonas fluorescens, Enterobacter aerogenes, Klebsiella oxytoca e Bacillus pumilus. Os PHAs produzidos por cada isolado foram extraídos com clorofórmio quente e os filmes poliméricos foram obtidos. Experimentos em frascos agitados (0 – 96 h) foram conduzidos para comparar a biomassa, glicídios redutores totais, pH e proteínas totais em meio mínimo contendo diferentes conteúdos de glicose e peptona. O suplemento com peptona demonstrou induzir o crescimento das linhagens, e o sobrenadante livre de células proveniente do cultivo de Pseudomonas fluorescens BMA-05 não apresentou acidificação em nenhuma das condições testadas. Na produção de polihidroxibutirato (avaliada após 24 h de incubação), utilizou-se o meio mínimo com diferentes fontes de nitrogênio: acetato de amônio, cloreto de amônio, nitrato de amônio, nitrato de sódio, sulfato de amônio, extrato de carne, extrato de levedura, glicina e peptona. O acúmulo máximo de P(3HB), detectado por espectrofotometria UV-visível, foi obtido quando P. fluorescens, E. aerogenes, K. oxytoca e B. pumilus foram cultivados no sulfato de amônio, cloreto de amônio, extrato de carne e nitrato de sódio, respectivamente. As observações em microscópio eletrônico de transmissão mostraram Pseudomonas fluorescens BMA-05 com grânulos eletronlucentes quando a linhagem foi cultivada na presença de sulfato de amônio ou nitrato de sódio. Embora nenhum grânulo tenha sido observado para as outras linhagens na presença do sulfato de amônio, as análises de cromatografia gasosa confirmaram a produção de P(3HB). Quando os testes de expressão de exoenzimas foram realizados, os resultados indicaram que as linhagens foram capazes de hidrolisar gelatina, amido e Tween 80, e B. pumilus causou hemólise em sangue de carneiro. Experimentos para a produção de biosurfactante/bioemulsificante indicam que E. aerogenes BMA-10 excretou um composto surfactante que colapsa na superfície hidrofóbica bem como emulsiona os hidrocarbonetos querosene, tolueno, óleo diesel e óleo de soja. O índice de emulsificação (E24) usando o sobrenadante livre de células desse microrganismo foi termalmente estável no tolueno, mas não em querosene. Resultados similares foram obtidos quando o pH do sobrenadante livre de células desse microrganismo foi ajustado para 2, 7 e 10, bem como quando concentrações diferentes de NaCl (2, 6 e 10 %) foram adicionadas às amostras. Tais resultados revelam o potencial de Enterobacter aerogenes BMA-10 como produtora de P(3HB), e o meio de cultura livre de suas células para finalidades emulsificantes em biotecnologia ambiental, bem como em biorremediação.

The presented diploma thesis is focused on preparation of nanoparticles and nanofibres with natural UV filters. Liposomes with encapsulated aqueous, ethanol and lipid extracts were prepared. Nanofibers from PHB containing lipid extract were prepared too. As a part of this work, an overview of natural sources with potential effects as UV filters were introduced. Moreover, nanoparticles and nanofibers and methods of their characterization were described. Size, polydisperse index and colloid stability of prepared nanoparticles were characterized via DLS. In experimental part aqueous, ethanol and lipid extracts were prepared from roasted coffee, green coffee and cascara. These extracts were spectrophotometrically characterized for the content of polyphenols, flavonoids, antioxidant activity, tannins and their SPF. Liposomes and liposomes containing PHB with these extracts were prepared and the encapsulation effectivity, short–term and long–term stability as well as SPF of nanoparticles were determined. Nanofibers from PHB containing lipid extracts were prepared via electrospinning and forcespinning methods. Prepared nanofibers were examined via FTIR–ATR. Antioxidant activity, short–term and long–term stability were determined spectrophotometrically. From selected nanoparticles, emulsions and gels were prepared and their SPF was also determined. Three types of emulsions with the best SPF were selected and tested on volunteers. Sedimentation stability of emulsions was tested by analytical centrifuge. Finally, cytotoxicity of selected nanoparticles and nanofibers was tested via MTT assay using human keratinocytes.

This dissertation work deals with the thermic and the mechanical behavior of plasticized bio-plastics and bio-composites for the 3D printing applications. The influence of plasticizer chemical structure on thermic and mechanical properties of plasticized polymeric blends from the poly-3-hydroxybutyrate and the poly lactic acid was investigated. Used plasticizers are based on derivative of citric acid. The influence of plasticizers on polymeric matrix and their compatibility was estimated by gear torque rate of melt mixer, respectively rate of plasticizer migration from the material during higher temperature. The plasticizer structure influence on the glass transition temperature and on the kinetics of crystallization of plasticized material was investigated by modulated differential scanning calorimetry. The behavior of material during 3D printing was also observed. Mechanical properties of printed samples, especially their elongation at break, were determined by tensile tests. The largest softening effect was observed using tributylcitrate plasticizer, where the glass temperature decreased by 35 °C and elongation at break increased by 150% compared to non-plasticized reference material. This plasticized polymeric blend showed also sufficient 3D printing properties and was used as the matrix for composites in the next part of this work. Composites were filled by kaolin, limestone, halloysit, fumed silica, talc, magnesium hydroxide and chopped flax fibers. Particle distribution in composites in dependence of used surface treatment of filler was observed by scanning electron microscopy. The influence of composite filler on rheological properties, crystallization kinetics and thermal stability of composites, was observed by viscometry and differential scanning calorimetry. Their mechanical properties and heat deflection temperature were observed on samples prepared by 3D print. Kaolin in composite material showed homogeneous particle distribution and insignificant nucleation effect and influence on thermic stability. Composite filled by kaolin also showed 18% smaller warping during 3D printing compared to non-filled reference. Consequently kaolin was evaluated as suitable inorganic filler for bioplastic composite intended for 3D print and this composite was used in the following part of this thesis. Method of mathematical prediction of Young's modulus was described for composite samples prepared by 3D print. Composites filled by one type of filler – kaolin, or limestone, resp. by combination of both fillers were investigated on the basis of the micromechanic Halpin-Tsai model modified by the semiempiric multiparametric Cerny's equation. Additive and combinational method of Young's modulus evaluation is used for composites with hybrid filling. Deflection of measured and theoretical Young's modulus value of composite filled with kaolin was decreased by established correction from 21% to 1% and for composites filled with limestone from 13% to 9%. In this manner it is possible to predict the Young's modulus of the samples prepared by 3D print.

Předložená diplomová práce se zabývá produkcí polyhydroxyalkanoátů (PHA) bakterií Cupriavidus necator H16. Cílem práce byla příprava, selekce a charakterizace mutantních kmenů schopných vyšší produkce PHA. V teoretické části byla zpracována literární rešerše zabývající se nejdůležitějšími typy PHA, bakterií Cupriavidus necator a způsoby indukce mutageneze. V experimentální části byly připraveny mutantní kmeny pomocí fyzikální a chemické mutageneze. Mutantní kmeny schopné nadprodukce PHA byly selektovány pomocí kultivace na minerálním médium s olejem. Pro další studium byly vybrány 4 mutantní kmeny schopné nadprodukce PHA. Tyto mutantní kmeny byly dále podrobeny biochemické charakterizaci. Byly naměřeny specifické aktivity vybraných intracelulárních enzymů včetně enzymů podílejících se na biosyntéze PHA. Také byla naměřena resistence mutantů vůči oxidačnímu stresu. Bylo zjištěno, že mutantní kmeny schopné nadprodukce PHA mají vyšší aktivity enzymů produkujících NADPH. NADPH je jeden z klíčových substrátů ovlivňujících směr toku acetyl-CoA metabolizmem. Vyšší intracelulární koncentrace NADPH parciálně inhibuje Krebsův cyklus a aktivuje akumulaci PHA. Aktivity acetoacetyl-CoA reduktázy a PHA syntázy, enzymů zapojených do syntézy PHA, těchto mutantů proto byly také vyšší stejně jako molekulová hmotnost připravených polymerů. Aplikace fyzikálních a chemických mutagenů je způsob, kterým lze připravit biotechnologicky perspektivní mutantní kmeny schopné nadprodukce PHA.

Presented work was focused on biodegradability of bacterial natural and modified polyesters and their composites. The first part of the work was focused on study of influence of PHA granules structure on their biodegradability using selected enzymes and influence of physiological conditions on PHA stability. Overall, tested polymer either in crytalinne or amorphous phase seems to be rezistent to attack of seleced hydrolytic enzymes such as lipases or proteases and is stable in simulated physiological fluids as well. Because of thies results, it is possible to use tested PHA materials in biomedical applications requiring rather resistant biomaterials. Second part of the work was focused on microbial degradation of modified PHA materials considering their potential environmental impact. Both mixed thermophillic culture originaly used in wastewater treatment plant and bacterial strain Delftia acidovorans were employed for biodegradation tests. Composites containing chlorine PHB and PHB films modified using plasticizers were tested. Films containing chlorine PHB cause inhibition of biomass growth to both tested cultures. The highest rate of degradation (31%) was observed in presence of bacterial culture with film containing 10% chlorine PHB. The results show that used microbial population is important factor affecting biodegradability.

The presented diploma thesis is focused on the preparation of new materials with antimicrobial effect. Liposomes and nanofibers from polyhydroxybutyrate containing clotrimazole and natural extracts with good antifungal and antioxidant effects were prepared. The theoretical part contains examples and short description of using nanoparticles and nanofibers in cosmetics and medicine and the description of plants which have positive and potential antimycotic effects. Moreover, methods for particles and fibers characterisation were shortly described. In the experimental part, natural water and lipid extracts were prepared and spectrophotometrically characterised for the content of polyphenols, flavonoids and the antioxidant activity. Liposomes and liposomes containtng PHB were prepared from selected extracts and the encapsulation effectivity, shortterm and longterm stability via determination of polyphenols were determined. Prepared particles were characterized with DLS method (size) and zeta- potential (stability). PHB nanofibers functionalised with selected lipid extracts and clotrimazole were prepared via electrospinning and forcespinning, and examined via FLIM and FTIR-ATR methods and spectrophotometry was used for antioxidant activity and release of active substances determination. Antifungal properties of prepared particles, extracts and fibers using the test system Candida glabrata were studied. Finally, cytotoxicity of selected samples was tested with MTT assay using human keratinocytes.