Le polyhydroxybutyrate (PHB) est un biopolymère intracellulaire synthétisé par fermentation bactérienne. Il présente de nombreux avantages (propriétés thermiques et mécaniques comparables à celles de polyoléfines, haute hydrophobie, biocompatibilité, biodégradabilité, industrialisable) et de nombreux inconvénients significatifs (mauvaise stabilité thermique et fragilité). Cette mauvaise stabilité est due à un phénomène de dégradation thermique proche du point de fusion du PHB. Ceci conduit à une réduction du poids moléculaire par un procédé de scission de chaîne aléatoire, modifiant alors irréversiblement les propriétés cristallines et rhéologiques de manière. Ainsi, les températures de fusion et de cristallisation du PHB diminuent drastiquement et la cinétique de cristallisation est ralentie. Par ailleurs, un second phénomène d'autonucléation intervient à proximité du point de fusion. En effet, une certaine quantité d'énergie est nécessaire pour effacer toute présence de résidus cristallins dans la matière à l’état fondu. Ces résidus peuvent agir comme nucléides pendant le processus de cristallisation et y influencer de manière significative la cinétique de cristallisation du PHB. Ce mémoire vise à montrer l'effet des processus de dégradation thermique et d’autonucléation du PHB sur sa cinétique de cristallisation. Pour cela, trois protocoles thermiques spécifiques ont été proposés, faisant varier la température maximum de chauffe (Th) et le temps de maintien à cette température (th) afin apporter une nouvelle approche de effet du traitement thermique sur la cristallisation, l’autonucléation, la dégradation thermique et la microstructure du PHB en utilisant respectivement la calorimétrie différentielle à balayage (DSC) dans des conditions cristallisation non-isotherme et isotherme, la diffraction de rayon X (WAXD), la spectroscopie infrarouge (FT-IR) et la microscopie optique. Th a été varié entre 167 et 200 °C et th entre 3 et 10 min. À Th ≥185°C, le phénomène de scission de chaine est le seul phénomène qui influence de cinétique de cristallisation alors qu’à Th < 180°C le processus de nucléation homogène est favorisé par la présence de résidus cristallins est prédomine sur le phénomène de dégradation. En ce qui concerne l'effet du temps de maintien, th, il a été mis en évidence le phénomène de dégradation thermique est sensible à la variation de ce paramètre, ce qui n’est pas le cas du processus d’autonucléation. Finalement, il a été montré que la morphologie cristalline est fortement affectée par les mécanismes de dégradation thermique et d'auto-nucléation.

Presented work is focused on study of polyhydroxybutyrate production in bacteria. In theoretical part short characterization of PHB was given and the most common representative of wide group of polyhydroxyalkanoates (PHA) were described. Then, production of PHB and copolymer P(HB-co-HV) in selected bacterial strains was experimentally proven. First, PHB production in Bacillus megaterium using synthetic medium was studied. The PHB content in cells was increased during cultivation under limiting conditions, despite low growth. Addition of ethanol into production media resulted in increased PHB synthesis as well as biomass production (21 % PHB of 1,8 g/l biomass). Further, BM medium containing 8 g/l glucose was tested. PHB production was more than 1 g/l at significant growth increase when compared with synthetic medium. The bacteria B.megaterium showed, except glucose, ability to utilize maltose and xylose. Another cultivations were tested with bacterial strain Azotobacter vinelandii, which is capable of copolymer P(HB-co-HV) synthesis. Maximal growth and copolymer content was reached on Burk's medium with 30 g/l of glucose. Addition of peroxide to growth medium influenced P(HB-co-HV) synthesis to 46 % of 2,6 g/l biomass. Bacteria A.vinelandii showed the best growth on maltose, even compared with glucose (54 % copolymer of biomass content). Finally, PHB production on industrial waste product – whey was monitored. Using Plackett-Burman design for statistical media optimization, the whey content was modified. B.megaterium grown on adjusted whey reached 0,5 g/l PHB, 32 % of cell's content.

Master of Science / Department of Chemistry / Ping Li / Biodegradable polyhydroxybutyrates (PHBs) produced by a wide range of bacteria have been considered as an ideal alternative to petroleum-based plastics. Two types of mechanistic probes have been synthesized in order to understand the mechanism of PHB synthases (PhaCs). The first type is oxo analogs in which the sulfur in the coenzyme A (CoA) thioester has been replaced with an oxygen atom. A series of 3-R-hydroxybutyryl oxo CoA analogs, (HB)[subscript]nOCoA (n = 1, 2 and 3), were synthesized chemoenzymatically in good yields. Two models involving covalent catalysis with Cys have been proposed for the chain elongation catalyzed by PhaCs. The first involves an active site composed of two monomers in which the growing hydroxybutyrate (HB) chain alternates between Cys on each monomer. The second involves noncovalent intermediates (HB)[subscript]nCoA (n ≥ 2). Here the substrate analog HBOCoA was successfully employed to trap the noncovalent intermediates in the reactions catalyzed by class III PhaC from Allochromatium Vinosum, which supports our preferred second mechanistic model. Furthermore, it is also the first time that a wild-type (wt) synthase was used to investigate the chain elongation models. The other type of mechanistic probes is 3-R-hydroxyalkyl CoA that was used to investigate the substrate specificity of PhaCs from different classes. Substrate availability has been a challenge to study PHB synthases in vitro. Starting with commercially available dimethyl S-malate, the intermediate S-ethyl 2-(oxiran-2-yl) acetate 23 was synthesized via a ring-opening reaction involving lactone 21 and trimethylsilyl iodide followed by an oxidation reaction involving silver oxide. The regiospecific ring-opening reaction of epoxide 23 with different organometallic reagents afforded a straightforward access to ethyl 3-R-hydroxybutanoates attached with a variety of side chains. The final CoA compounds were obtained through the thiotransesterification reaction between corresponding benzenethioesters and the thiol group in CoA. This synthetic approach provides a new avenue to modifications of alkyl groups in 3-R-hydroxyalkyl CoA in an efficient manner.

The goal of this thesis was to summarize basic options of fiber spinning and manufacturing and their usability in industry. Methods of nanofiber spinning are described in the teoretical part, alongside the materials used in experimental part. The experimental part was focused on creating of workflow of defined nanofibers in laboratory enviroment, the possible modification of nanofibers with aditives. The release of aditives from different nanofibers into different enviroments was measured.

This diploma thesis deals with the applicability of thermal analysis in the influence of stress factors on microorganisms. The main aim of the presented work was to design and implement a series of experiments to thest the usability of advanced methods of thermal analysis in the characterization of microorganisms. Two bacterial strains were used as model organisms: Cupriavidus necator H16 and its mutant strain Cupriavidus necator PHB-4, which differs in the ability to produce polyhydroxyalkanoates. The basic technique used thermogravimetric analysis (TGA), which was optimized and applied in the study of water transport, behavior and determination of intracellular water content with respect to the influence of the presence of intracellular PHB granules. The results of this method were subsequently supported by TEM photos.

Polyhydroxyalkanoates (PHAs) are a type of biologically-produced plastic known for their biocompatibility and biodegradability. They have the potential to replace petroleum-based plastics as an environmentally-friendly alternative. This is beneficial because the release of plastics into environments such as the ocean and the buildup of plastics in landfills are major concerns facing society today. Currently, however, PHAs are significantly more expensive than their petroleum-based counterparts. This is largely due to the cost of carbon sources and of extracting the bioplastics from bacteria. The goal of these studies was to examine replacing traditional carbon sources used in PHA production like sugar and oils with sustainable carbon sources and to improve extraction procedures by inducing secretion of PHAs in bacteria. A few sustainable carbon sources were examined for use in PHA production. First, studies focused on the conversion of food waste into PHAs were reviewed. It was shown that utilizing food wastes as carbon sources may be a viable approach to producing PHAs. A second carbon source examined was methanol. A novel isolate of Methylobacterium that demonstrated the ability to produce PHAs from methanol was identified. A system of secreting PHAs that was constructed using synthetic biological engineering approach was introduced to this isolate. This secretion system was not shown to improve extraction of PHAs in Methylobacterium in its current form.

Azotobacter vinelandii contains an iron-regulatory small RNA ArrF whose expression is dependent upon the levels of iron and ferric uptake regulator. The deletion of ArrF-encoding gene resulted in a 300old increase in the production of polyhydroxybutyrate (PHB), a polymer of industrial importance. This ∆arrF mutant exhibited wild-type growth and growth-associated PHB production. Limited iron and aeration elevated the PHB production in the mutant as well as wild type. SDS-PAGE and MALDI-MS/MS revealed the overexpression of acetyl-CoA reductase, a phbBAC operon enzyme and the proteins that would alleviate the stress due to PHB accumulation in the ∆arrF mutant. Real-time RT-PCR revealed that phbR, phbB, phbA and phbC were upregulated in the mutant. Increased levels of activator PhbR in the mutant elevates the expression of phbB, phbA and phbC, resulting in the PHB overproduction. The proteins differentially expressed in the ∆arrF mutant were determined by gel-based proteomics and confirmed by real time RT-PCR. 6-phosphogluconolactonase that involve in the production of NADPH and acetyl-CoA, was upregulated, while the proteins involved in the TCA cycle that consumes acetyl-CoA were downregulated. Heat-shock proteins such as HSP20 and GroEL were overexpressed in the mutant. In addition, antioxidant proteins such as Fe-containing supeoxide dismutase (FeSOD), a putative oxidoreductase with unknown function, alkyl hydroperoxide reductase, flavorprotein WrbA and cysteine synthase were also upregulated, indicating that the PHB accumulation is highly stressful to the cells. Upregulated in the ∆arrF mutant were acetyl-CoA carboxylase, flagellin, and adenylate kinase. Among the genes upregulated in the ∆arrF mutant, sodB gene coding for Fe-superoxide dismutase and phbF gene encoding PHB synthesis regulator appears to be negatively regulated by small RNA ArrF in an antisense mechanism. However, all the TCA cycle genes were downregulated in the ∆arrF mutant. In addition to the TCA cycles enzyme, glutamate synthetase, elongation factor-Tu, iron ABC transporter, and major outer membrane porin OprF were downregulated in the ∆arrF mutant. Based on the results, it is concluded that several factors are responsible for the overproduction of PHB polymer in the ∆arrF mutant and one of which is the direct effect of small RNA ArrF on the expression of PhbF .

Des polyacides lactiques hydroxy téléchélique de masses molaires variées ont été synthétisés par polymérisation par ouverture de cycle du LLA en présence de 1,4 -butane diol. Des polyhydroxybutyrate hydroxy téléchéliques ont été préparés transesterification du PHB et du 1,4-butanediol catalysée par l'acide p-toluène sulfonique .Ces oligomères ont été utilisés pour préparer des polyesters amides fonctionnalisés acrylates ou mercaptans. Dans une approche parallèle, du PCL multi acrylate a été préparé avec succès par copolymérisation par ouverture de cycle de méthacrylate de glycidyle avec du caprolactone. Des copolymères (GMA -co- CL) ayant des masses molaire et de la fonctionnalité variables, ont été préparés en modifiant les paramètres de la réaction tels que le catalyseur, la nature du co-amorceur et le rapport des stœchiométriques des différents réactifs. Les polymères multi acrylates ont été copolymérisés principalement avec l'HEMA sous irradiation UV pour obtenir des réseaux PLA, PHB et PCL segmentés. Les mécanismes de dégradation de ces réseaux ont été étudiés en examinant particulièrement les décompositions des liaisons uréthanes et esters. Des dépolymérisations du PHEMA ont été détectées avec TGA -FTIR à plus haute température (450°C). Pour les réseaux à base de PCL, les propriétés thermo -mécaniques ont été étudiées. Le résultat montre que les phases riches en PCL ont une bonne compatibilité avec le poly HEMA. Les modules caoutchoutiques et l’étendue des températures des zones d’amortissement peuvent être contrôlées en fonction des paramètres réactionnels. Les Polymères multifonctionnels Thiols du PCL, PHA et PLA ont été utilisés pour faire croître des chaines méthacrylates et construire des polymères de type étoile / Hydroxyl telechelic polylactic acids of various molecular weights were synthesized by ring opening polymerization in the presence of LLA and 1,4-butanediol. Telechelic hydroxy polyhydroxybutyrate were prepared transesterification of PHB and 1,4- butanediol catalyzed by p- toluene sulfonic acid. These oligomers were used to prepare polyesteramides functionalised acrylates or mercaptans. In a parallel approach, the PCL multi acrylate was successfully prepared by ring-opening copolymerization of glycidyl methacrylate with caprolactone. Copolymers (GMA-co-CL) with variable molar masses and functionality were prepared by changing the reaction parameters such as catalyst, the nature of the co-initiator and the ratio of different stoichiometric reagents. Multi-acrylated polymers were copolymerized with HEMA under UV irradiation to obtain PLA, PHB and PCL segmented networks. Degradation mechanisms of these networks have been studied by examining particular decomposition of urethane bonds and esters. Depolymerization of the PHEMA was detected with TGA -FTIR at higher temperature (450°C). For PCL based networks, the thermo- mechanical properties were studied. The result shows that the PCL-rich phases have good compatibility with poly HEMA. The rubber and the working temperature range of the damping zones modules can be controlled as a function of reaction parameters. Multi mercapto functionalized polymers with PCL, PLA and PHA segments were used to grow methacrylate polymer chains and build star type

A produção de PHAs é limitada devido ao alto custo da fonte de carbono para á produção. No Brasil, o uso de xilose uma fonte de carbono abundante no bagaço de cana é uma alternativa. Neste estudo o catabolismo de xilose em B. sacchari foi estudado para explorar seu potencial para a produção de PHB. Primeiro a organização do operon de xilose foi descrita e foi demostrado que a superexpressão de xylAB melhoro a velocidade máxima de crescimento assim como o teor de acumulo de PHB. Depois foi identificado o fenômeno de repressão catabólica, o qual foi abolido a traves da superexpressão dos genes xylE xylAB. Finalmente foi criado um set de plasmídeos induzíveis para fazer engenharia no consume de xilose em B. sacchari. A superexpressão de xylR permitiu que B. sacchari atinge a velocidade máxima de crescimento mais alta reportada e o melhor fator de conversão de xilose a PHB. Foi concluído então que a superexpressão de xylAB e xylR ajudam a melhorar a velocidade máxima de crescimento e a capacidade de acumulo de PHB usando xylose como fonte de carbono em B. sacchari. / Polyhydroxyalkanoate production is limited by the high production cost of carbon sources. The use of cheap carbon sources like xylose is an alternative to address this issue. In this work we aimed to understand and engineer xylose catabolism in B. sacchari, a bacteria isolated in Brazil to exploit its potential for producing PHB from renewable sources. Initially, we described organization of xylose assimilation genes and demonstrated that xylAB overexpression is an efficient strategy to improve B. sacchari growth rate and production of PHB using xylose as sole carbon source, achieving the highest conversion rate and titer described. Then we identified B. sacchari sequential preference for different sugars (glucose>arabinose>xylose) and overexpress xylE-xylAB to abolish this preference. Finally we created a set of inducible vectors and use them to engineer xylose metabolism. Overexpression of xylR, allowed B. sacchari cells to achieve the highest growth rate and PHB conversion factor and yield reported using xylose as a sole carbon source. Finally, we conclude that overexpression of xylAB and xylR genes improved growth rate, conversion factor and yield when PHB is produced using xylose as carbon source in B. sacchari.

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.