Halomonas species as a source of poly(3-hydroxybutyrate). Preparation of hydrophilic macroporous polymer scaffolds
Sammanfattning: This thesis presents studies on moderately halophilic bacteria belonging to the family Halomonadaceae that accumulate polyhydroxybutyrate (PHB) in high amounts when grown under conditions of nitrogen limitation. PHB is the simplest homopolymer belonging to the class of polyhydroxyalkanoates (PHAs), the biopolyesters that can biodegraded in the environment to carbon dioxide and water and are hence attractive substitutes for fossil-derived plastics. Halomonas andesensis was isolated from a hypersaline lake in the Andean region of Bolivia, and was identified as a novel species by phenotypic and genotypic characterization. The organism utilizes different carbon sources such as acetate, L-arabinose, cellobiose, D-fructose, galactose, D-gluconic acid, D-glucose, L-lactic acid, maltose, myo-inositol, propionic acid, raffinose, L-rhamnose, sucrose, starch and D-xylose, respectively, for growth. Halomonas boliviensis was also earlier isolated from the same environment. Cultivation of H. boliviensis in shake flasks with glucose and sucrose, respectively, as carbon sources, resulted in a cell dry weight (CDW) of 8.6 g l-1 and PHB content of 52.7 wt%. Even with dried molasses as the carbon source, the CDW and PHB content were 9.4 g l-1 and 43.9 wt%, respectively. Glutamate was shown to be an important nitrogen source that could replace the more complex yeast extract for growing the bacteria, while its depletion facilitated the PHB accumulation in the cells. When H. boliviensis was grown under fed-batch conditions using glucose as the carbon source and glutamate as the nitrogen source, the cell mass increased to 44 g l-1 with a PHB content of 81 wt%. Production of PHB was also investigated by fed batch cultivation of Halomonas meridiana, obtained from a culture collection. Using glucose and maltose, respectively, as carbon sources, a cell dry weight of 27 g l-1 was obtained in 21 hours with a maximum PHB content of 65 wt%. The ability of the halophiles to grow on different carbon sources makes possible the use of alternative and cheap carbon sources for the production of the biopolyester. Analysis of the genome of H. boliviensis revealed that a total of 160 genes are related to the transport and metabolism of carbohydrates, and most of them were obtained from other bacteria by horizontal gene transfer. Furthermore, glycolysis and gluconeogenesis in H. boliviensis guide a high level of polymorphism for the enzymes involved in the transformation of different carbon sources. Such a variety of enzymes in the pathways explains the versatility for the choice of substrate by the bacterium. Similar metabolic evolutionary traits may also be observed in other members of the family Halomonadaceae owing to the common phenotypic features among them. Due to their excellent biodegradability and biocompatibility, PHAs are attractive for use as carriers for tissue engineering and other biomedical applications. PHB is hydrophobic and brittle, and requires modification to promote optimal cell growth. PHB extracted from H. boliviensis was used to prepare scaffolds by thermally induced phase separation of the polymer solution in dioxane. The scaffolds prepared at -12 C and below, followed by removal of the solvent, were macroporous. A new method for modification of the scaffolds was developed involving treatment with the enzyme lipase to generate carbonyl groups that were subsequently covalently modified with glucosamine and gelatin, respectively. The modified scaffolds had a lower molecular size and melting temperature, and significantly higher water content. Human embryonic fibroblasts were grown on the modified materials, which were shown to promote cell adhesion, proliferation, and spreading. These results open up new possibilities of coupling other molecules to give the scaffolds the necessary features for different types of cells.
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