Este trabalho teve por objetivo avaliar o potencial de 39 isolados bacterianos para produção de polihidroxialcanoatos contendo monômeros de cadeia média (PHA|MCL). Os isolados foram incapazes de utilizar eficientemente amido, lactose ou sacarose. O desempenho na produção de PHA a partir de glicose ou frutose pelos isolados foi inferior à linhagem de referência (Pseudomonas sp. LFM046). Muitos dos isolados apresentaram bom desempenho na conversão de glicerol em PHAMCL. Pela primeira vez, foram detectadas bactérias capazes de produzir PHAMCL a partir de xilose. Com base no sequenciamento do rDNA 16S, estes isolados foram identificados como pertencentes ao gênero Pseudomonas. Cultivos em biorreator, resultaram em uma biomassa de aproximadamente 6 g/L, contendo cerca de 11 a 14% de PHA. A superexpressão dos genes responsáveis pelo catabolismo de xilose (xylAB) em Escherichia coli não levou a melhora no consumo de xilose ou produção de PHAMCL. / This work aimed to evaluate the potential of 39 bacterial isolates to produce polyhydroxyalkanoates containing medium-chain monomers (PHAMCL). The isolates were unable to efficiently utilize starch, lactose or sucrose. The performance of the PHA production from glucose or fructose by the isolates was lower than the reference strain (Pseudomonas sp. LFM046). Many isolates showed good performance in the conversion of glycerol into PHAMCL. For the first time, bacterial isolates capable of producing PHAMCL from xylose were detected. Based on the sequencing of 16S rDNA, these isolates were identified as belonging to the genus Pseudomonas. Bioreactor cultures resulted in a biomass of approximately 6 g/L, containing about 11 to 14% PHA. Overexpression of the genes responsible for the catabolism of xylose (xylAB) in Escherichia coli did not led to improved xylose consumption or production of PHAMCL.

Os polihidroxialcanoatos constituem um grupo de poliésteres acumulados por inúmeras bactérias na forma de grânulos intracelulares, que podem representar até 80% da massa seca celular. No presente trabalho, foi avaliado o comportamento de diferentes linhagens quanto a capacidade de metabolizarem ácidos graxos livres e óleo de soja para a produção de biopolímeros e, posteriormente, em biorreator, algumas linhagens selvagens e recombinantes foram testadas na presença ou não de um inibidor da b-oxidação. Experimentos em frascos agitados, mostraram valores de teor de PHA em até cerca de 70% na biomassa total com cerca de 5% de monômeros de cadeia média permitindo selecionar as linhagens Cupriavidus necator e Burkholderia cepacia para os ensaios seguintes. Experimentos em biorreator mostraram que, as variáveis manipuláveis quanto ao fluxo de óleo de soja, de co-substratos e o inibidor de b-oxidação não influenciaram, significativamente, no rendimento de P3HB/3HAmcl formado quando utilizado Burkholderia cepacia IPT-048, entretanto, contribuíram na síntese de 3HV. Durante os ensaios, o aumento do número de cópias do gene phaB em Cupriavidus necator, aparentemente, não contribuíram para o aumento do rendimento de 3HHx, porém, outras variáveis manipuláveis deverão ser propostas para a confirmação. / The poly-3-hydroxyalkanoates (PHA) are a group of polyesters accumulated for several bacteria in the intracellular granule form, that can represent up to 80% of the dry mass cellular. The main advantage of the biopolymers on the synthetic materials is its degradation in the environment. Recycable raw materials can be used as carbon sources for the production biodegradable polymer. Some polymers have appeared in literature having mechanical characteristic considered appropriate in such a way for use in packings (flexible and covering films), how base for controlled release of asset to be applied in the pharmacological-medical and foods area. The P3HB-co-3HAmcl are 3-hydroxybutyrate copolymers and 3-hydroxyacyl of 6 or more carbon atoms which has aroused interest for to present intermediate properties between HB and HAmcl, having taken care of the requirements for diverse applications. Some strains are distinguished, however, assays in bottles agitated with the Cupriavidus sp. and Burkholderia sp. strains, using as substratum greasy free acid and soy oil, had led the promising results. In the present work, first, was evaluated the behavior of different strains (Cupriavidus necator DSM545 IPT-026, C. necator H16 IPT-027, Pseudomonas sp. IPT-066, Burkholderia cepacia IPT-048 and B. sacchari IPT-189) how much to metabolizer capacity of free fatty acids and soybean oil for the production of biopolymers and, later, in tank bioreactor some wild strains and recombinant had been tested together or not of an inhibitor of the boxidation (acid acrylic). Experiments in bottles with agitation, had shown values of PHA in about 70% of the total biomass with about 5% monomers chain average allowing to select the Burkholderia cepacia IPT-048 and Cupriavidus necator DSM545 strains for the following assays. Experiments in tank bioreactor had shown that, the interactions between the substratum, co-substratum and the inhibitor of boxidation had influenced in the amount of formed P3HB/3HAmcl when used the Burkholderia cepacia IPT-048 strains, contributing for the synthesis of 3HV and 3HHx. When used only the soybean oil (5g/L) was gotten an amount of HHx with about 6 mol% with Burkholderia cepacia strains. In this same condition was verified that after some determined period also proved monomer HV in an amount of 0.20 mol%. Adding the acid acrylic (0.18 g/L) were gotten 1.50 mol% of HHx, however, differently of the previous condition the amount of HV varied of 0.91 - 2.77 mol%. A conclusion for the condition soybean oil and acrylic acid is that, the strains is using the acrylic fatty acid as carbon source, or either, the formation of acrylil-CoA with consequent formation of chains with 5 carbons. Similar results had been gotten when analyzed extracted polymer, or either, after the stage of extraction with chloroform was verified that the amount varied of 1.60 - 2.14 mol%. When added to a cosubstratum in the phase of accumulation (caproic fatty acid) and absence of the acrylic acid, it did not have the increase of HHx in relation to the previous variable, as much that, the amount of monomer reached about 0.50 mol%. In this condition, the presence of the acrylic fatty acid also did not contribute for the HV synthesis, not having been observed monomers during the assays. In the following phase, in which we work with Cupriavidus necator and a recombinant (Cupriavidus necator::phaB) strains, was verified that the gotten amount of HHx had been similar, about 0.30 mol%. Our hypothesis, we believe that the increase of the number of copies of the gene phaB could contribute for the increase of the carbon flow in the direction to raise the amount of 3HB how much of other monomers. As in the assays in bottles with agitation substrat caproic and caprylic fatty acids they had been distinguished also when availabled 2g/L and the soybean oil of in a concentration of 5g/L, we analyze the strains recombinant with soybean oil and caprylic fatty acid with 5 g/L. The insistence in the use of the caprylic fatty acid was based on the fact of the possibility of strains to oxidate part of fatty acids for the production of cells and energy and, at the moment that had the nitrogen limitation, to begin the accumulation phase dividing two carbons for the production of acetyl-CoA and the remain of the chain was stored in the form of 3HHx. The caprylic fatty acid disponibility it did not have the monomer attainment, therefore, Cupriavidus necator and Cupriavidus necator::phaB had its replyed inhibited capacity by the substratum. In the added soybean oil bottles with and without the acrylic fatty acid the behavior it was the opposite to the observed with caprylic fatty acid. During the incubation period, for both the strains, the amount of PHA, and consequently, the percentage of 3HB in the dry mass cellular was of 22.95% (100 mol%) and 25.90% (100 mol%) in 28ª and 24ª hours, respectively. In this condition, in the recombinant strains, also was observed the presence of HHx between 32ª and 49ª hour with a maxim amount of 1.10 mol%. From the results it can be concluded that the Burkholderia cepacia is considered a promising strains for the attainment of HHx and HV allowing that soybean oil with and without the acrylic fatty acid, respectively. The increase in the number of copies of the gene phaB in Cupriavidus necator apparently did not contribute with the increase of 3HHx efficiency however, other handling variables will be proposed for confirmation.

The aim of this diploma thesis was study of the isolation of polyhydroxyalkanoates using a commercial surfactant and selected biosurfactants, which were sophorolipids, coconut soap, lecithin, lauryl glucoside, coco glucoside and cocamidopropyl betaine. PHA was isolated from Schlegelella thermodepolymerans DSM 15344, where the amount of NaCl in the production medium was first optimized. The molecular weight of the isolated polymer was analyzed by SEC-MALS. The largest structural changes were recorded for coconut soap. Possible impurities in the isolated polymer were analyzed by infrared spectrometry (ATR-FTIR). The least protein contamination of the polymer was recorded with coconut soap. From the above biosurfactants, coconut soap, lauryl glucoside and coco glucoside were selected, because the highest purity of PHA was obtained. However, coconut soap had the greatest potential. The isolation temperature and surfactant concentration were optimized for selected biosurfactants. The best results were obtained at 90 °C and a concentration of 5 g/L. Furthermore, the versatility of the isolation method was tested using selected biosurfactants on various thermophilic microorganisms, which were Chelatococcus composti DSM 101465, Schlegelella thermodepolymerans DSM 15264, Tepidimonas taiwanensis LMG 22826 and Aneurinibacillus thermoaerophilus H1.

This master’s thesis deals with the study of composting of polyhydroxybutyrate (PHB) and polylactic acid (PLA). The experimental part focuses on the composting test based on the IS/ISO 20200 standard, in which biopolymers were degraded within 8 weeks. The advantage of the composting test was the unique use of bags for polymer granules, which simplified the handling of samples. The placement of the polymers ensured an even load of compost, which simulated the landfill environment, which also made this experiment exceptional. The use of different methods of analysis ensured the investigation of the influence of compost on the degradation of polymers, but also the influence of polymers on the compost microbiome. In the compost environment all of the studied polymers degraded. After 8 weeks the weight loss of approximately 40 % was detected in PHB. Amorphous PLA degraded completely in the 5th week of experiment, while semicrystalline PLA lost approximately 70 % of its weight in 8 weeks. Using SEM images and SEC analysis, we conclude that PHB degradation was predominantly biological. Degradation of PLA samples was found to be predominantly abiotic. Analysis of compost samples revealed that the enzymatic activity of esterases increased in composts with polymers compared to compost without polymers. The effect of polymers on the microbiome in compost was analyzed using Biolog EcoPlates™. Compost with PHB showed a high ability to adapt to various substrates and microbiome expanded during composting. In the case of a sample with semicrystalline PLA, it was found that in the last week of composting, the ability of microorganisms to adapt and use substrates decreased, which indicates a possible negative effect of this polymer on compost development.

This master thesis deals with the development of a minibioreactor for microbial biotechnologies. The AlgaTox system – an analytical photobioreactor from BVT Technologies – was the default unit of the new device. The working volume of the new minibioreactor is in the range of 4 to 8 ml. The minibioreactor was composed of a minithermostat, a reaction vessel, an oxygen electrode, a temperature and pH probe, accessories for the supply of air to the liquid and an insertion for the transport of liquid from / to the reactor. The functionality of the assembled device and its characteristics were measured. Furthermore, an operating procedure for the decontamination process using a hydrogen peroxide mist was developed for the new equipment. An operating procedure for culturing microorganisms in a minibioreactor was also set up. Pilot cultivations of Halomonas halophila were demonstrated in the prepared equipment. In these cultivations, three assemblies for air supply to the liquid were tested. However, none of the assemblies was able to provide a sufficient supply of oxygen to cell culture – the dissolved oxygen value always dropped to 0%. For the further development, a new assembly was designed for the supply of air to the liquid - an aeration ring from a membrane tube.

This master’s thesis focuses on biodegradation of polyhydroxybutyrate (PHB) and polylactic acid (PLA). The theoretical part discusses an origin, properties and applications of investigated biopolyesters, and so the enzymology of their biodegradation. The experimental part deals with biodegradation of these polymers in liquid medium using several pure thermophilic bacteial strains and controlled composting of these bioplastics. Amongst six tested thermophilic bacterial strains only one showed PHB-biodegradation activity – strain Schlegelella thermodepolymerans. No degradation degree of amorphous or semi-crystalline PLA was observed. Mainly disintegration of both forms of PLA articles was observed in compost environment, thus the abiotic mechanism of its decomposition was indicated. After 4 weeks of composting, the relative weight loss of 99 % and 63 % was detected in amorphous and semi-crystalline PLA respectively. On the contrary, the weight loss of PHB after 4 weeks of composting reached 36 %, moreover, a half decrease of molar mass was observed using SEC. The surface erosive mechanism of PHB-biodegradation was stated using SEM. By monitoring of esterase, lipase and protease activities, no influence on the compost by polymer presence was concluded at statistical significance.

This diploma thesis deals with the isolation and characterization of copolymer P(3HB-co-4HB). The teoretical part was prepared as a literature search which describe polyhydroxyalkanoates in general, their structure, synthesis, degradation and isolation. Furthermore, copolymers containing 4HB units are mentioned in this thesis and there is also no mentioned of the biosynthesis and biodegradation of copolymer P(3HB-co-4HB). The first aim of this diploma thesis was to find out which solvent is the best for copolymer extraction and then characterize obtained copolymer P(3HB-co-4HB). The isolated copolymer was characterized by gas chromatography, SEC-MALS, thermal analysis and SEM. In the second part of the thesis, release of model biologically active substance from the PHA films was studied. Rhodamine 6G dye was selected for the simulation, which was used to stain the copolymer films and the P(3HB) films. It was observed that film prepared from P(3HB-co-4HB) copolymer released entrapped substance much faster than film made from P3HB homopolymer, and, in addition, the copolymer was substantially more susceptible to enzyme degradation.

Polyhydroxyalkanoates (PHA) are microbial storage polyesters that represent a renewable and environmentally friendly alternative to petrochemical plastics. However, their production and use are severely disadvantaged by the high production cost. The use of extremophilic PHA producers is one of the ways to reduce the cost of PHA production. Extremophiles bring numerous advantages resulting from the high robustness of the process against microbial contamination. In this doctoral thesis, attention was focused on the study of PHA production using selected halophilic and thermophilic microorganisms. Representatives of the genus Halomonas were mainly from public collections of microorganisms. Two promising PHA producers on waste frying oil were identified, namely Halomonas hydrothermalis and Halomonas neptunia. Both strains achieved good PHA yields in flask experiments. With the addition of suitable structural precursors, they were also able to produce copolymers with interesting material properties. However, in the proposed thesis, the main emphasis was placed on the study of PHA production using thermophilic microorganisms. As a part of the work, the isolation of thermophilic PHA producers from various thermophilic consortia (active sludge, compost, etc.) was performed. During isolations experiments, an original isolation procedure was designed using changes in osmotic pressure, the so-called osmoselection. Dozens of promising thermophilic PHA producers were obtained thanks to this original approach. They were taxonomically classified using 16S rRNA and tested for production potential. The most promising PHA producer was the isolate which was classified as Aneurinibacillus sp. H1. This bacterium is able to utilize a variety of substrates, including waste glycerol, to produce PHA. Even more important is the capability of synthesizing copolymers with a high content of 4-hydroxybutyrate. The monomer composition of the PHA copolymer and thus the material properties of the prepared copolymer can be controlled by suitable adjustment of the cultivation conditions. The prepared copolymer P(3HB-co-4HB) has unique properties and the great application potential in numerous high-end applications, for example in the field of health care, food industry or cosmetics.

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.