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Biotechnological production of polyhydroxyalkanoates by thermophiles
Kouřilová, Xenie ; Patáková, Petra (referee) ; Koutný, Marek (referee) ; Obruča, Stanislav (advisor)
Polyhydroxyalkanoates (PHAs) are microbial biopolymers that may provide a partial alternative to petrochemically produced plastics. Their main advantages are biodegradability, biocompatibility, and the possibility of production from renewable sources. However, the cost of their production is still higher than that of petroleum-based polymers. To increase the competitiveness of biotechnological processes, the concept of Next Generation Industrial Biotechnology (NGIB) has been introduced by other authors. This concept relies on the use of extremophilic microorganisms. When extremophiles are implemented in fermentation processes, the robustness of these technologies is increased and at the same time sterility requirements can be reduced. In the context of PHA production using extremophiles, a subset of halophilic microorganisms is relatively well mapped. Significantly less information is available on another interesting category, namely thermophiles. In line with the idea of NGIB, it is the production of PHAs by thermophilic bacteria that is the focus of this thesis. Attention is paid mainly to the genera Caldimonas, Rubrobacter, and Tepidimonas. For several tested representatives of these genera, the parameters investigated were the optimal cultivation temperature, suitable carbon substrate and the ability to produce copolymers. Based on this basic screening parameters, the most promising producers were selected and subjected to further experiments. Representatives of the genus Rubrobacter have the advantage of being gram-positive non-sporulating bacteria, as the risk of contamination of the isolated polymer by pyrogenic lipopolysaccharides present in the cell wall of gram-negative microorganisms is eliminated. The bacterial strain Tepidimonas taiwanensis LMG 22826T was able to produce high yields of biomass and PHA on a mixture of glucose and fructose substrate. Using grape pomace extract, almost identical values to those obtained on pure substrates were achieved. The thermophile Caldimonas thermodepolymerans DSM 15344, originally named Schlegelella thermodepolymerans, is a very promising PHA producer on xylose-based substrates. These substrates can be, for example, hydrolysates of lignocellulosic materials, which represent a sustainable source of carbon. Their suitability for the cultivation of C. thermodepolymerans was tested on model hydrolysates composed of pure carbohydrates. To approximate real samples, the effect of potential microbial inhibitors present in lignocellulosic hydrolysates was also investigated. An innovative isolation protocol for the recovery of PHAs based on osmotic stressing of thermophilic and halophilic microorganisms under elevated temperature with the addition of a low concentration surfactant solution was also developed as part of the thesis.
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Evolutionary and genetic engineering of bacterial producers of polyhydroxyalkanoates
Nováčková, Ivana ; Patáková, Petra (referee) ; Koutný, Marek (referee) ; Obruča, Stanislav (advisor)
This doctoral thesis deals with the topic of evolutionary and genetic engineering of polyhydroxyalkanoates (PHA) producing bacteria. Apart from these topics, the issue of biotechnological production of PHA on model hydrolysates of lignocellulosic biomass with the use of extremophilic microorganisms is also studied, as well as the development of an alternative method of PHA isolation. The themes were freely linked to previous experiments and reflected the currently solved projects in a working group. Doctoral thesis is prepared in the form of a commented discussion of published works, which are part of it in the form of appendices. Evolutionary engineering was mainly applied to the model PHA producing bacterial strain Cupriavidus necator H16. By adaptation to levulinic acid, isolates producing copolymer P(3HB-co-3HV) with a higher content of the 3HV fraction were obtained, which leads to improved properties of the polymer for further processing. As well as culture growth also the amount of total PHA in the biomass was higher. By long-term adaptation of the same strain to osmotic stress and the presence of copper ions, the isolates which are characterized in the second publication, were obtained. Based on obtained data, it was possible to observe differences in the adaptation process, where the adaptation to osmotic stress was gradual, while a significant step in the increase of biomass and PHA signaling faster adaptation was observed for copper. Based on the analyses, the significant role of PHA in the adaptation of the C. necator H16 strain to the tested stressors was discussed, it did not consist only in the increase in the amount of polymer in the biomass, but also in enhancement of whole PHA cycle, which also leads to an increase of the pool of monomeric units showing protective functions. By adaptation to -captolactone, a unique precursor of 4HB, the copolymer P(3HB-co-4HB) was obtained. The properties of this copolymer are again more favorable than of the homopolymer P(3HB), even with a low content of 4HB, which we also achieved in a laboratory bioreactor. A further increase in the 4HB fraction could be achieved using deletion mutants with the absence of relevant genes, which is discussed more in the text. The production of PHA on models of lignocellulosic biomass hydrolysates originating from, for example, the food industry was tested in combination with the use of extremophile producers, when the preference of the contained monosaccharides (hexoses, pentoses) for individual producers was discussed. For the purpose to get closer to real hydrolysates, the resistance of the strains to relevant potential microbial inhibitors was also tested. The susceptibility of halophilic and thermophilic PHA producers to osmotic stress was used in the development of an alternative isolation approach that would reduce the economic and ecological burden of the process compared to standard extraction using chlorinated solvents. Application of SDS detergent at low concentrations while simultaneously exposing the cells to higher temperatures led to the gain of high purity polymer without loss of yield. The recycling process of used SDS is also a possibility.
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