A 13C-Análise de Fluxos Metabólicos (13C-MFA) tornou-se uma técnica de alta precisão para estimar fluxos metabólicos e obter informações importantes sobre o metabolismo. Este método consiste em procedimentos experimentais, técnicas de medição e em cálculos para análise de dados. Neste contexto, os grupos de pesquisa de engenharia metabólica necessitam de ferramentas computacionais precisas e adequadas aos seus objetos de estudo. No presente trabalho, foi construída uma ferramenta computacional na plataforma MATLAB que executa cálculos de 13C-MFA, com balanços de metabólitos e cumômeros. Além disso, um módulo para estimar os fluxos metabólicos e um módulo para quantificar as incertezas das estimativas também foram implementados. O programa foi validado com dados presentes na literatura e aplicado a estudos de caso. Na estimação de fluxos de Pseudomonas sp. LFM046, identificou-se que esse micro-organismo possivelmente utiliza a Via das Pentoses em conjunto com a Via Entner-Doudoroff para a biossíntese de Polihidroxialcanoato (PHA). No design ótimo de experimentos para uma rede genérica de Pseudomonas, identificou-se a glicose marcada no átomo cinco como um substrato que permitirá determinar o fluxo na Via das Pentoses com menor incerteza. / 13C-Metabolic Flux Analysis (13C-MFA) has become a high-precision technique to estimate metabolic fluxes and get insights into metabolism. This method consists of experimental procedures, measurement techniques and data analysis calculations. In this context, metabolic engineering research groups demand accurate and suitable computational tools to perform the calculations. A computational tool was implemented in MATLAB platform that performs 13C-MFA calculation, using metabolite and cumomer balances, as well as a module to estimate the fluxes and a module to quantify their uncertainty. The program was validated with some classical cases from literature. From the flux estimates of Pseudomonas sp. LFM046, it was identified that the microorganism possibly uses the Pentose Phosphate Pathway along with the Entner-Doudoroff Pathway for Polyhydroxyalkanoate (PHA) biosynthesis. From the optimal experimental design for a generic Pseudomonas network, it was possible to conclude that glucose labeled at atom five is the best option to determine the flux in the Pentose Phosphate Pathway with smaller uncertainty.

Les matériaux polymères ont de nombreux avantages comme la légèreté, le coût, la formabilité… mais sont aussi à l’origine de certains problèmes environnementaux actuels. La substitution des polymères conventionnels d’origine pétrochimique et non biodégradables par des polymères biosourcés et biodégradables tels que le polylactide (PLA) ou les polyhydroxyalcanoates (PHA) peut apparaître comme une alternative crédible. Cependant, un des freins à leur développement demeure la faible connaissance de la durée de vie de ces biopolymères lorsqu’ils sont exposés à différents types d’environnements, en particulier en milieu marin si l’on considère une application marine. Dans le cadre de cette étude, il a donc été entrepris de suivre le vieillissement naturel et le vieillissement accéléré du PLA et des PHA en milieu marin dans l’objectif d’appréhender les mécanismes et les cinétiques de dégradation. Dans le but de découpler les effets liés à l’eau, aux sels minéraux et aux microorganismes, les échantillons ont été immergés dans différentes conditions, i.e. en eau distillée, en eau de mer naturelle et en eau de mer filtrée et renouvelée à différentes températures. L’influence du milieu sur la dégradation de ces biopolymères est analysée et discutée, de même que l’influence de la géométrie des pièces (films de différentes épaisseurs, éprouvettes ou fibres). Enfin, la prédiction de la durée de vie de ces polymères a été envisagée par l’intermédiaire de deux approches, conduisant à une estimation de leur durabilité dans un milieu bien défini et au regard d’une propriété donnée. / Pollution of nature by plastics is a major environmental problem and better management of the lifetime of polymers is a major challenge for the future. In recent years, bio-based and biodegradable polymers, such as polylactide (PLA), or polyhydroxyalkanoates (PHA) have appeared as an alternative solution in order to solve these problems. One of the limits remains the relative lack of knowledge of their lifetime and degradation behaviour in aqueous environments, and more specifically in the marine environment. In this study natural and accelerated ageing tests were performed under several conditions, distilled water, filtered and renewed seawater and natural seawater, at different temperatures, in order to decouple enzymatic and hydrolytic mechanisms. The aim of this study is to establish a baseline on degradation mechanisms and kinetics, in order to make lifetime predictions of biopolymer behaviour in seawater.Degradation phenomena have been identified. Biodegradation tests were also performed in a marine environment by following the release of CO2. Then, lifetime predictions of the properties of these biopolymers at seawater temperature were made using two different approaches.

The thesis deals with bioinformatics analysis, the aim of which was to find a suitable producer of PHA for new generation industrial biotechnologies from the collection of found thermophilic bacteria. Part of experiments was the finding of several thermophilic bacteria based on the similarity of the protein sequence of the phaC gene of the bacterium Cupriavidus necator. The next part of thesis was a literature search of the abilities of these thermophilic bacteria focused on culture conditions and the spectrum of usable substrates. Subsequently, five bacteria were selected for use in NGBI based on the information obtained. Freely available databases were used during the experimental work, and evolutionary analysis were performed in MEGA X and Operon-mapper. Rubrobacter xylanophilus with collection number DSM 9941 was selected from the collection of bacterial strains as the most promising PHA producer for NGIB. The high culture temperature of up to 70 ° C and a large amount of utilized carbohydrate substrates were considered decisive. An interesting result of the analysis was to find the gene sequences of two classes of PHA synthase – I. and III. class, as for a single bacterial strain from the entire collection. Additional genes linked to PHA metabolism were found in genome analysis.

Polyhydroxyalkanoater (PHA) är en grupp av polymerer som är biobaserade, biologiskt nedbrytbara och som kan uppvisa varierande fysiokemiska egenskaper. Således har PHA potential att ersätta konventionell och fossilbaserad plast så som polypropylen (PP) och polyetentereftalat (PET).  PHA framställas genom mikrobiologisk fermentering av organiskt material och innesluter sig i bakteriernas celler som sfäriska lagringsdepåer när bakterier matas med en kolkälla. PHAt kan sedan extraheras ut från bakterierna med hjälp av lösningsmedel, en metod som ger höga utvinningsnivåer men som kräver stora mängder lösningsmedel. Ska metoden vara hållbar och ha industriell relevans är det nödvändigt att lösningsmedelsåtervinning tillämpas.   Industriell framställning av PHA sker idag med rena bakteriekulturer som matas med odlade sockerrika grödor. Detta leder till att konkurrens om markanvändning samt vatten- och energiresurser för främst livsmedelsproduktion uppstår. Samtidigt skapas restströmmar från industri och samhälle innehållande organiskt material och blandade bakteriekulturer vilka kan utnyttjas för att producera PHA.   I detta arbete har PHA-processen modellerats i processimuleringsprogrammet CHEMCAD där data på restströmmar från skogsindustrin utnyttjas för PHA-produktion. I modellen har uppströmsprocesserna fermentering, anrikning, PHA-ackumulering och avvattning samt nedströmsprocesserna lösningsmedelsextraktion och separation inkluderats. Nedströmsprocesserna har modellerats för två fall. Ett fall där extraktion gjorts med 100 % torr biomassa (extraktionssystem 1) samt ett fall där extraktion gjorts med våt biomassa (extraktionssystem 2). Därtill implementerades intern värmeväxling vilket optimerades genom införandet av pinchanalys.   Målet med projektet var att upprätta en energianalys av PHA processen, identifiera energibehovet för de enskilda PHA-processerna, analysera om lösningsmedelsextraktion görs mest energieffektivt med våt eller torr biomassa samt att utvärdera hur intern värmeväxling kan bidra till att energieffektivisera PHA-processen.  Resultaten som erhölls visade att behovet av extern värme kunde minska från 49 till 13 MJ/ kg PHA och kylbehovet från 43 till 11 MJ/ kg PHA efter införandet av intern värmeväxling för extraktionssystem 1. När extraktion utfördes enligt extraktionssystem 2 minskade det externa kylbehovet från 98 till 48 MJ/kg PHA och det externa värmebehovet från 60,9 till 8,1 MJ/ kg PHA. I jämförelse med det totala specifika värme- och kylbehovet var det totala specifika elbehovet för PHA-processen lågt vilket kan ses som positivt då el är högvärdig och dyr energi. När extraktion utfördes enligt extraktionssystem 1 var det totala specifika elbehovet 3,55 MJ/ kg PHA och för extraktionssystem 2 var motsvarande siffra 4,1 MJ/ kg PHA. Störst elbehov uppstod i processen för luftning av PHA-ackumuleringstanken vilket krävde 2,8 MJ/kg PHA. / Polyhydroxyalkanoates (PHAs) is a group of biobased and biodegradable polymers which can exhibit flexible physicochemical properties. Thus, PHA has the potential to replace conventional and fossil-based plastic such as polypropylene (PP) and polyethylene terephthalate (PET).  PHA is produced through microbiological fermentation by organic matter and is stored in the cells of bacteria as spherical storage depots when the bacteria are feed by a carbon source. The PHA can then be extracted from the bacteria using solvent, a method resulting in high yields of PHA but on the other hand large quantities of solvent is required and if the method will be sustainable and have industrial relevance it is necessary to apply solvent recycling in the system.  Today industrial production of PHA takes place using pure bacterial cultures and cultivated sugar-rich crops. This contributes to a competition of land use and water and energy resources, mainly for food production. At the same time, waste streams are generated from industry and society containing organic material and mixed bacterial cultures which can be used to produced PHA.  In this work the process for PHA production has been modelled in the process simulation program CHEMCAD using data from waste streams produced from forest industry. In the model the following upstream processes has been included; acidogenic fermentation, enrichment, PHA-accumulation, dewatering and the downstream processes solvent extraction and separation. Moreover, the downstream processes have been modelled for two cases; one case where extraction takes place using 100 % dry biomass (extraction system 1) and one case where extraction takes place using wet biomass (extraction system 2). In addition, internal heat exchanging was implemented which was optimized through the introduction of pinch analysis.   The aim of the project was to establish an energy analysis of the PHA process, identify the energy needs of the individual PHA processes, analyse whether solvent extraction is made most energy efficient using wet or dry biomass and to evaluate how internal heat exchanging can contribute to make to the PHA process more energy efficient.   The results obtained showed that the need for external heating and cooling could decrease from 49 to 13 MJ/ kg PHA and 43 till 11 MJ/ kg PHA after the implementation of internal heat exchanging for extraction system 1. When extraction was performed according to extraction system 2 the external cooling demand was reduced from 98 to 48 MJ/kg PHA and the externa heating demand was reduced from 60,9 to 8,1 MJ/ kg PHA. In comparison with the total specific heating and cooling demand, the total specific electricity demand for the PHA process was low, which can be seen as positive as electricity is high-value and expensive energy. When extraction was performed according to extraction system 1, the total specific electricity requirement was 3,55 MJ/ kg PHA and for extraction system 2 the corresponding value was 4,1 MJ/ kg PHA. The greatest electricity demand arose in the process of aerating the PHA accumulation reactor with 2,8 MJ/kg PHA.

The presented dissertation thesis deals with the polymer polyhydroxybutyrate and other biopolymers as a basic building block for the construction of micro- and nanoscopic structures and materials used in food and cosmetics. In the theoretical part, current literary review is prepared to introduce the basics of this application field. The practical part of the work is composed of three blocks developed during the doctoral study. In the first and most important part are summarized comments to the academic and patent outputs, where among the academic ones it is possible to find two peer-reviewed articles dealing with the electrostatic and wet spinning of PHB and properties of prepared materials. The patent outputs consist of several accepted and applied projects, which summarize results on both PHB spinning methods, but also on general approaches enabling the processing of PHB into forms enabling many applications in food and cosmetics. Second part was focused on the patented composition of the UV protection cream based on the prepared nanoscopic and micro- morphologies of PHB. The third block summarizes results focused predominantly on the electrostatic spinning of PHB and other biopolymers. Finally, a short chapter containing a brief description of projects that were in a way related to the dissertation topic, but rather practical development work in Central Tanzania and West Africa, which draw on knowledge and contacts gained during studies at FCH BUT Brno.

The aim of this work is to study microbial production of polyhydroxyalkanoates (PHA). Theoretical part is focused on production of PHA using microoganisms and transgenic plants. Bacterial strain Cupriavidus necator H16 was used for laboratory production of PHA. Various waste oils were used as sole carbon and energy source. Salt of propionic adic and 1-propanol were used as intermediate for 3-hydroxyvalerate monomer (3HV) unit. Incorporation of 3HV to polymer can improve material features of PHA. The major part of experimental work was focused to study influence of aeration (concentration of dissolved oxygen) to bacterial growth, selected metabolic pathways and formation of PHA. Furthermore, influence of aeration to monomer composition of polymer was evaluated. According to experimental conclusion of this work it was approved dependance between aeration and monomer composition of PHA. Moreover, it was approved that higher concentration of oxygen supports bacterial growth and influences PHA content in cells. In addition, NADPH is one of the substrates influencing flux of acetyl-CoA throughout the metabolism; higher intracellular concentration of NADPH inhibits TCA cycle and enhances accumulation of PHA in cells. For this reason, specific enzymatic aktivity of several selected intracelular enzymes were measured, including those enzymes which can generate NADPH.

The master‘s thesis is focused on the study of biodegradation of polyhydroxyalkanoates, namely polymer polyhydroxybutyrate. The first part of the thesis is focused on the study of biodegradation of polyhydroxybutyrate in the form of crystalline granules of PHB and PHB films using selected species of microorganisms from bacteria, yeasts and fungi. As a representative of bacteria was chosen microorganism Delftia acidorovans, as yeast was selected Aureobasidium pullulans and Aspergillus fumigatus as fungi. PHB depolymerase activity was measured employing turbidemtiric method with suspension of PHB granules as substrate. The results showed that D. acidorovans can partially degrade PHB. On the contrary A. pullulans cannot effectively degrade PHB. The most significant degradation ability revealed A. fumigatus, which was able to degrade PHB completely. Extracellular enzymes excreted by these microorganisms when cultivated on PHB materials as sole carbon sources were analyzed by SDS-PAGE. The second part of the thesis deals with the biodegradation of PHB in the form of PHB film, PHB hardened foil and PHB Nanoul fabric using standard composting test. Semi-solid cultivation showed positive results. In the interval from 14 days to two months were all forms of the PHB completely biodegraded. With semi-solid cultivation was also studied biodegradation rate of the polyurethane elastomeric films which were modified by partial replacement of polyester polyol by PHB. The test samples were prepared using PHB from Sigma and the PHB samples prepared at the Faculty of chemistry VUT. Samples with different concentrations of the dispersed PHB (1 %, 5 % and 10 %) in the polyurethane were also object of the study. At the end of the cultivation (after 2 months) were measured mechanical properties in tension of the material, then efficiency of biodegradation by gravimetric analysis and modification of the material surface by microscopic analysis.

Suitable skidding trail layout is important in sustainable forestry system, thus can minimise the damages to trees and economical losses. In a cooperation between Mendel University in Brno and Kasetsart University in Bangkok a potential use of photogrammetry based skidding trail layout was evaluated. Certain plots of teak plantation in Thong Pha Phum were captured with and further processed in Agisoft Metashape software. On obtained orthophoto single trees were distinguished and understory trees for thinning from below were identified on Weighted Voronoi diagram base. On Kernel Density Estimation base a tree density estimation raster output were created. Considering irregular teak spacing a subjective approach for skidding trail layout design was applied, with emphasis to shorten skidding distances and reducing the possible damages to remaining trees and soils.

Since their conception, plastics have been a dominant product due to the versatility of use, and readily available raw materials. However, the production and consumption of synthetic plastics have continued to increase over the years, leading to a significant rise in plastic waste and its associated environmental impacts, such as the detrimental effects plastic waste has on ecosystems, including the presence of toxic microplastics and risks to marine life, are becoming the focus of criticism. Moreover, the contribution of plastic production to global warming through the usage of petroleum as a raw material cannot be understated. Solutions are being sought after to reduce the impact of this plastic waste, with one such solution being the replacement of the raw materials involved in plastic production. If a biologically degradable plastic product can be produced, then even if the dangers associated with plastic waste cannot be removed, they can be reduced. One method is the production of plastics using polyhydroxyalkanoates (PHA) as a replacement as these are shown to have similar properties as synthetic plastics while being 100% degradable. PHA production has existed since the 1960’s, but the process has always been too expensive to be a viable alternative to the cheaper petroleum-based products. A reduction of the production costs is needed for PHA to be an economically viable alternative, and there are two areas that contain the highest costs; The usage of expensive monocultural bacteria, and specially crafted carbon sources used to feed these bacteria to stimulate PHA accumulation. By switching to a multi-cultural bacterium and using readily available organic carbon sources can the costs of production be brought down, allowing the prospective biological plastics a chance to compete in the plastic market.  This thesis focuses on the usage of a multi-cultural bacterium collected from the water purification plant of a papermill, and the filtrate from the hydrothermal carbonisation (HTC) of sludge as the organic carbon source as materials in the production of PHA. The aim of the thesis is to investigate how successful PHA accumulation can occur using these two readily available waste products, and if successful to analyse the characteristics of any biological plastic that could be produced. The trials were performed in a cylindrical tank; one benchmark trial using acetic acid as the carbon source to be used as a reference for successful PHA accumulation, and five trials investigating the PHA accumulation that occurred using the HTC filtrate as the carbon source. The trials were analysed by extraction, TGA, FT-IR, TOC, and SS. During the trials pH, temperature and concentration of diffused oxygen was monitored. The trials indicated that the accumulation of PHA was possible, with various results. The most successful accumulation was observed in trial I-1, where PHA reached 12.45%, only slightly lower than the benchmark trials accumulation of 13.6%. Trials I-2 and I-3 also showed potential for high PHA content according to FTIR analysis. However, trials F-1 and F-2 failed to accumulate any PHA, and the inhibiting factors behind this were not fully understood. Possible reasons included high ammonia levels, incompatible bacteria, or imbalanced nutrient ratios. Various extraction methods were attempted to isolate the polymers found within the biomass for use in plastic production. While one extraction method was successful and used to analyse the PHA content found in the biomass, the amount of polymer that was extracted via this method was too small for use in plastic production, and as such the characteristic investigation of the plastic was unable to be completed. Overall, the study identified successful PHA accumulation in some trials but faced challenges and uncertainties regarding inhibiting factors and extraction methods. Future improvements should focus on standardizing experimental conditions and optimizing extraction techniques for better results.

Lead contamination threatens human life and the environment. The biological reduction of Pb(II) to metallic Pb is an attractive solution for Pb(II) pollution. Delftia acidovorans, Azonexus caeni, and Comamonas testosteroni were isolated and studied for their capabilities to utilize Pb(II) as a terminal electron acceptor. D. acidovorans strain Pb11 and A. caeni strain Pb2 cultures showed a 5.2- and 8.1-fold growth at 10.0 mg-Pb(II)/L in 3 d, respectively. Petroleum-based plastics are another emergent environmental concern. Polyhydroxyalkanoates (PHAs), a sustainable alternative to conventional plastics, are biodegradable polymers produced by PHA accumulators under autotrophic or heterotrophic conditions. In this thesis, the growth and enrichment of PHA accumulators, such as Plasticicumulans acidivorans and Cupriavidus necator, were investigated as they can accumulate 90% of their cell weight as PHA. An energetic model was developed to calculate theoretical PHA yields. The true autotrophic and heterotrophic PHB yields were estimated as 2.97 (g_PHB/ 〖mol〗_(H_2 )) and 0.66 (g_PHB/ g_acetate), respectively. Moreover, the growth of C. necator was investigated in lab-scale experiments under various autotrophic, heterotrophic, and mixotrophic conditions. When C. necator was cultivated in two-stage systems, high optical densities were attained in less than 24 h. In addition, a mathematical model for the competition between PHA and non-PHA accumulators in the feast-famine enrichments was developed. The calibrated and validated model for P. acidivorans suggested that microbial diversity in mixed cultures impacted the enrichment process. Another aspect of this thesis was to propose an innovative method for enriching PHA accumulators in mixed cultures. By applying autotrophic and autotrophic-heterotrophic enrichment strategies, C. necator dominated the mixed cultures (> 90%) in less than five days. Based on this thesis findings, it can be concluded that biotechnology applications in Pb(II) remediation and PHA production could reduce the severe impacts of Pb contamination, petrochemical plastics, and climate change due to elevated CO2 levels. / Thesis / Doctor of Philosophy (PhD) / This thesis aimed to provide sustainable biotechnological solutions to three environmental challenges: lead contamination, petroleum-based plastics, and elevated CO2 levels in the atmosphere. Certain metal-reducing bacteria can grow by consuming toxic Pb(II) ions from aqueous environments and thus reduce their toxicity. Furthermore, various microorganisms can store biodegradable polymers, known as polyhydroxyalkanoates (PHAs), in their cells. The stored PHA polymers can be extracted and processed to produce biodegradable plastics. PHA accumulators can produce significant amounts of PHA by utilizing organic substrates or CO2. Therefore, PHA-based plastics can reduce environmental deterioration due to non-degradable plastics and elevated CO2 levels. Lab-scale experiments and mathematical modeling can provide a better understanding of the growth and enrichment of PHA accumulators in engineered PHA-production systems. Research findings in this thesis will allow cost-effective and sustainable production of biodegradable plastics from organic wastes and flue gas.