Ett av världens största miljöproblem är plastnedskräpning. På många platser kan spår av mänsklig närvaro ses i form av skräp av plast. År 2011 tillverkades det 280 miljoner ton plast, det motsvarar ungefär 28 000 Eiffeltorn. I Sverige förbrukades år 2010 ungefär 880 000 ton plast. I Asien produceras ungefär 50 % av all världens plast och Kina står för cirka hälften av detta. Nordamerika och Europa står för cirka 40 % av världens plastproduktion. Resterande produktion av plast är fördelat på Afrika och Sydamerika. Kommersiell plast är uppbyggd av små enheter kallade polymerer. Polymerer är i sin tur uppbyggda av ännu mindre enheter som kallas monomerer. Dessa monomerer är i dagsläget framställda av petroleum (råolja/mineralolja). Ungefär 4 % av världens oljekonsumtion går åt som råvara till att producera plast och lika mycket olja används som bränsle i tillverkningsprocessen. Begreppet bioplast är en hel familj av material som är biologiskt nedbrytbar, biobaserade eller bådadera. Det är dock inte en självklarhet att bioplaster besitter båda egenskaperna. PHA-plast är biobaserad och biologisk nedbrytbar, vilket är anledningen till att den står i fokus under detta examensarbete. För produktion av PHA-plast används en trestegsprocess, vilket innefattar ett fermenteringssteg, ett selektionssteg och ett ackumuleringssteg. Sist sker en extraktion för att frigöra PHA- plasten från det övriga organiska materialet. Syftet med det här examensarbetet är att med hjälp av framställning av bioplast främja miljön, vilket en anläggning som producerar bioplast skulle göra eftersom en del av den fossiloljebaserade plasten skulle kunna bytas ut mot bioplasten PHA. Större framställningsmöjligheter av bioplast i Värmland skulle medföra ett ökat intresse av en produktionsanläggning. Målet är att inventera olika industrier runt om i Värmland, i första hand matindustrier och skogsindustrier, och utreda deras processavloppsvattens potential att producera VFA. I detta examensarbete har fermenteringsförsök genomförts satsvis på processavloppsvatten från OLW, Barilla (Wasa), Skoghall, Gruvön och Rottneros. Försöken visar deras potential att producera VFA. Experimenten utfördes med ett konstant pH på 6 och varierande uppehållstid. Resultaten visade att OLW och Barilla har bäst potential till VFA-produktion med 4500 mg/l respektive 1610 mg/l. Spädning av OLWs och Barillas processavloppsvatten visade sig vara en gynnsam åtgärd, då VFA-produktionen ökade snabbare i jämförelse med de tester som utfördes vid icke-spädning. Dock erhölls inte lika stor totalmängd av VFA. Det är dock bättre att producera en större mängd VFA och på så vis låta processen ta längre tid. Vid fortsatta experiment rekommenderas att göra ytterligare försök på OLW och Barillas processavloppsvatten då de visade bäst potential till VFA-produktion. / One of the biggest environmental problems is the plastic littering. In many places traces of human presence is seen in the form of plastic littering. In the year 2011, 280 million tons of plastic was produced, which represents about 28 000 Eiffel Towers. In Sweden, about 880 000 tons of plastic a year is consumed, according to figures from 2010. Approximately 50 % of all the world's plastics are produced In Asia and China accounts for about half of it. North America and Europe account for about 40% of the world's plastic production. The remaining production is distributed between Africa and South America. Commercial plastic is made from small units called polymers. A polymer consists of smaller units called monomers. In present, these monomers are produced out of petroleum (crude oil/ mineral oil). Approximately 4% of the world’s oil consumption is spent as raw material to produce plastic and the same amount of oil is used as fuel in the plastic production process. The term bio-plastic is used for a family of materials which are biodegradable, bio- based or both. However, it is not given that bioplastics do possess both properties. PHA plastics are both bio based and biodegradable, which is why it is the focus for this thesis. Production of PHA plastic is a three-step process comprising a fermentation step, a selection step, and an accumulation stage. Finally, there is an extraction to release the PHA plastic from the organic material. The aim of this thesis is to aid the production of bioplastics in order to lessen the environmental load of plastics. The more bioplastic that can be produced, the greater the interest of a bioplastic-producing plant in Värmland. The goal is to make an inventory of industries around Värmland, primarily food industries and forest industries, and to quantify the potential of their process wastewaters to produce VFA. In this thesis, fermentation experiments conducted batch-wise was performed with process wastewater from OLW, Barilla (Wasa), Skoghall, Gruvön and Rottneros. The experiments showed the wastewaters potential to produce VFA. The experiments were performed with a constant pH of 6 and with varying residence time. The results showed that OLW and Barilla has the highest potential for VFA production with 4500 mg/l and 1610 mg/l, respectively. Dilution of OLWs and Barillas process water turned out to be favorable, as the VFA production increased rapidly in comparison with those tests that were conducted under non-dilution. The total production of VFA, however, was not as high. In further experiments, it is recommended to make another attempt at the OLWs and Barillas process wastewater since they showed the best potential for VFA production.

Polihidroxialcanoatos (PHA) são poliésteres acumulados por diversas bactérias a partir de fontes renováveis e são termoplásticos, biodegradáveis e biocompatíveis. A variabilidade da composição monomérica de PHA determina suas propriedades mecânicas e permite seu uso em diversas aplicações. A PHA sintase é a enzima responsável pela polimerização do PHA. O objetivo deste trabalho foi à busca por genes codificadores desta enzima, construção e avaliação de recombinantes portando tais genes. Inicialmente buscaram-se novos genes de PHA sintase a partir de clones de uma biblioteca metagenômica previamente detectados por PCR como positivos para algum tipo de PHA sintase. Posteriormente buscou-se PHA sintases de classe III e construiram-se recombinantes de Pseudomonas sp e B. sacchari pela introdução de genes de C. vinosum (phaECCv). Nas duas recombinantes, os genes inseridos foram capazes de aumentar a fração de 3HHx em relação a linhagem selvagem, quando se utilizou glicose e hexanoato como fontes de carbono. / Polyhydroxyalkanoates (PHA) are polyesters accumulated from renewable sources by several bacteria and are thermoplastic, biodegradable and biocompatible. The variability of the PHA monomer composition determines its mechanical properties and allows their use in many applications. PHA synthase is the enzyme responsible for the polymerization of PHA. The objective was to search for genes encoding this enzyme, construction and the assessment of recombinant bacteria carrying such genes. Initially a screening of PHA synthase genes was made from a metagenomic library clones previously identified as positive by PCR for any type of PHA synthase. Later a search for class III PHA synthases was made as a construction of a recombinant Pseudomonas sp and B. sacchari by introducing genes of C. vinosum (phaECCv). In the two recombinant strains, the genes inserted were able to increase the fraction of 3HHx compared to the wild strain when glucose and hexanoate was used as carbon sources.

Polyhydroxyalkanoate (PHA) is a cellular storage polymer with similar properties to some plastics when extracted from the cell and acts as a renewable and biodegradable plastic alternative. The objective of this research was to identify thermophilic PHA producing microorganisms and characterize their PHA accumulation, by which the process may become more competitive to current petroleum-based plastic production. This study characterizes growth and PHA production of Thermus thermophilus HB8 and three strains of T. filiformis (MOK, NGM and WRT), while simultaneously performing a genetic analysis for the presence of a phaC gene that encodes the PHA synthase. The results from the genetic analyses, together with attempts to produce PHA in T. thermophilus HB8 and T. filiformis strain (WRT, NGM and MOK) infer an absence of PHA accumulation. Contrary to our expectations, Thermus sp. is not a strong candidate for the thermophilic production of PHA.

Polyhydroxyalkanoate (PHA) is a cellular storage polymer with similar properties to some plastics when extracted from the cell and acts as a renewable and biodegradable plastic alternative. The objective of this research was to identify thermophilic PHA producing microorganisms and characterize their PHA accumulation, by which the process may become more competitive to current petroleum-based plastic production. This study characterizes growth and PHA production of Thermus thermophilus HB8 and three strains of T. filiformis (MOK, NGM and WRT), while simultaneously performing a genetic analysis for the presence of a phaC gene that encodes the PHA synthase. The results from the genetic analyses, together with attempts to produce PHA in T. thermophilus HB8 and T. filiformis strain (WRT, NGM and MOK) infer an absence of PHA accumulation. Contrary to our expectations, Thermus sp. is not a strong candidate for the thermophilic production of PHA.

Polihidroxialcanoatos (PHA) são poliésteres acumulados por diversas bactérias a partir de fontes renováveis e são termoplásticos, biodegradáveis e biocompatíveis. A variabilidade da composição monomérica de PHA determina suas propriedades mecânicas e permite seu uso em diversas aplicações. A PHA sintase é a enzima responsável pela polimerização do PHA. O objetivo deste trabalho foi à busca por genes codificadores desta enzima, construção e avaliação de recombinantes portando tais genes. Inicialmente buscaram-se novos genes de PHA sintase a partir de clones de uma biblioteca metagenômica previamente detectados por PCR como positivos para algum tipo de PHA sintase. Posteriormente buscou-se PHA sintases de classe III e construiram-se recombinantes de Pseudomonas sp e B. sacchari pela introdução de genes de C. vinosum (phaECCv). Nas duas recombinantes, os genes inseridos foram capazes de aumentar a fração de 3HHx em relação a linhagem selvagem, quando se utilizou glicose e hexanoato como fontes de carbono. / Polyhydroxyalkanoates (PHA) are polyesters accumulated from renewable sources by several bacteria and are thermoplastic, biodegradable and biocompatible. The variability of the PHA monomer composition determines its mechanical properties and allows their use in many applications. PHA synthase is the enzyme responsible for the polymerization of PHA. The objective was to search for genes encoding this enzyme, construction and the assessment of recombinant bacteria carrying such genes. Initially a screening of PHA synthase genes was made from a metagenomic library clones previously identified as positive by PCR for any type of PHA synthase. Later a search for class III PHA synthases was made as a construction of a recombinant Pseudomonas sp and B. sacchari by introducing genes of C. vinosum (phaECCv). In the two recombinant strains, the genes inserted were able to increase the fraction of 3HHx compared to the wild strain when glucose and hexanoate was used as carbon sources.

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.

Les poly(hydroxyalcanoate)s (PHAs) sont des polyesters biodégradables et biocompatibles d'intérêt pour leur application dans le domaine biomédical ou en substitution des plastiques dérivés de l'industrie pétrolière. La synthèse des PHAs par polymérisation par ouverture de cycle (ROP) de monomères cycliques, les β-lactones, permet un bon contrôle de la masse molaire, de la microstructure et de la fonctionnalité des polymères. Les propriétés des polyesters peuvent être modulées par l'utilisation de différents monomères. Depuis quelques décennies, différents PHAs ont été synthétisés, notamment le poly(hydroxybutyrate) (PHB) et les poly(malolactonate d'alkyle)s (PMLARs). Ces travaux ont été étendus à la ROP d'une nouvelle famille de β-lactones, les 4- alcoxyméthlylène-β-propiolatones (BPLORs). Ainsi, différents systèmes catalytiques à base d'yttrium offrent des (co)polymères de tacticité (syndiotactique, atactique ou isotactique) et de topologie (copolymères à blocs, alternés ou aléatoires) originales. La stéréosélectivité des systèmes catalytiques mis en œuvre est inédite dans le contexte scientifique actuel. En effet, la nature des substituants des ligands ancillaires des complexes d'yttrium permet de moduler la microstructure et donc les propriétés des PHAs obtenus. / Poly(hydroxyalkanoate)s (PHAs) are biodegradable and biocompatible polyesters of interest for their application in the biomedical field or as alternative to plastics derived from the petroleum industry. The synthesis of PHAs by ring-opening polymerization (ROP) of cyclic monomers, β-lactones, enables a good control of the molar mass, the microstructure and the functionality of the polymers. The properties of the polyesters can be tuned by the using different monomers. In recent decades, various PHAs have been synthesized, in particular poly(hydroxybutyrate) (PHB) and poly(alkyl malolactonate)s (PMLARs). This work has been extended to the ROP a new family of β-lactones, namely 4-alkoxymethlylene-β-propiolatones (BPLORs). Thus, various yttrium-based catalyst systems provide (co)polymers with original tacticity (syndiotactic, atactic or isotactic) and topology (block, alternating or random copolymers). The stereoselectivity of the catalytic systems implemented is unprecedented in the current scientific context. Indeed, the nature of the substituants on the ancillary ligand of the yttrium complexes enables to tune the microstructure and therefore the properties of the resulting PHAs.

No description available.

This diploma thesis is focused on isolation and identification of thermophilic microorganisms capable of production of polyhydroxyalkanoates (PHA) in the sample of activated sludge from wastewater treatment. 6 culture samples were isolated from activated sludge by means of cultivation technics and methods of molecular biology. They were closer specified by comparing nucleotide sequences of 16S-rRNA gene and assigned to bacterial genus Anoxybacillus. The production of PHA by this genus was not reported in literature so far. Samples were confirmed to contain phaC gene that codes the enzyme PHA-synthase and they also gave a positive response to staining colonies with Nile red, what refers to presence of intracellular lipidic structures. However, the PHA production by isolates was not successful. The reason should be an inappropriate production medium or conditions. The positive phenotype result of Nile red dyeing was probably achieved by production of huge amount of lipids by bacterial cells that provides similar fluorescence than PHA granules.

This diploma thesis focuses on the molecular characterization of selected PHA producers. Within this work, the PHA producing thermophilic isolates originating from the samples of activated sludge and compost were identified and characterized using molecular biological methods. By sequencing the 16S rRNA gene, the thermophilic isolates were identified and taxonomically classified into the Firmicutes bacterial phylum. In these bacterial isolates, the ability to produce PHA at the genotype level was determined by conventional PCR detection of the phaC gene encoding PHA synthase, which is a key enzyme in PHA biosynthesis. Class I, II and IV PHA synthases were detected in most of the isolated bacteria, wherein class I and II PHA synthases are not characteristic for these bacterial genera. The largest proportion of isolates was identified for the species of thermophilic bacterium Aneurinibacillus thermoaerophilus, in which class IV PHA synthase was detected. In the second part of the diploma thesis, the RT-qPCR method was implemented to study the expression of selected genes of the bacterium Cupriavidus necator H16 involved in PHA metabolism. As part of the implementation of this method, PCR-based detection of selected genes was optimized and quantification of genes using real-time PCR was performed. The tested method included steps of RNA isolation, cDNA synthesis and quantification of gene segments for which the critical points of the method were determined based on the obtained data.