Production of Ectoines and Poly(3-Hydroxybutyrate): High Cell-Density Cultivation of Halomonas Boliviensis

Sammanfattning: This thesis reports a study involving the use of Halomonas boliviensis, a moderate halophile, for production of biopolyester poly(3-hydroxybutyrate) (PHB) and compatible solutes ectoine and hydroxyectoine through high cell-density cultivation comprising fed-batch cultures. PHB is a biodegradable plastic produced by bacteria as energy reserve and can potentially be used as a replacement of fossil based plastic. Ectoines comprise osmoprotectants and find use as stabilizers of cells and biomolecules. High PHB content and volumetric productivity were achieved by fed-batch culture of H. boliviensis using an optimized medium with glucose as carbon source. Cell growth was favored in the initial phase of cultivation by providing sufficient nutrients to the fermentor. Nitrogen and phosphorus feed was then limited to trigger PHB accumulation. This resulted in a cell dry weight, PHB content and volumetric productivity of 44 g L-1, 81 wt% of the cell dry weight and 1.1 g L-1 h-1, respectively. Further optimization of the feeding strategy was studied in two-stage fed-batch cultivation. Prolonged feeding of nutrients during the first stage and subsequent nutrient limitation increased the cell dry weight to 91 g L-1 but lower PHB content than in previous studies. However, the volumetric productivities achieved were slightly improved and are comparable to the highest values reported so far. Two-step fed-batch fermentation with medium exchange was used to optimize the cell growth and production of ectoines by Halomonas boliviensis. A mathematical method was applied to determine the optimal conditions for biomass and ectoine production. A two-fed-batch process was then applied for obtaining high cell mass as a first step, and transferring the cells to a second fed-batch reactor with fresh medium at higher salinity to initiate ectoines synthesis. This strategy resulted in high ectoines content of 27.8 wt% and an overall ectoines volumetric productivity of 10 g L−1 d−1. Ectoines were then released from the cells by “bacterial milking” process by subjecting the cells to osmotic downshock without significant loss of cell viability. Further recultivation of the cells allowed them to re-synthesize the ectoine in a new cycle in shorter time. This resulted in an overall volumetric productivity of 11.1 g L−1 d−1, the highest reported so far for fed-batch cultivation for the production of ectoines. A process for simultaneous co-production of ectoines and PHB by H. boliviensis using two-step fed-batch cultivations with medium exchange was developed in order to reduce the production cost of both products. After obtaining high cell-density under optimum growth conditions in fed-batch cultivation, sodium chloride concentration was increased to induce ectoine synthesis, as well as limitation of nutrients was applied to achieve PHB accumulation during the second cultivation step. The result was ectoine content and volumetric productivity of 7.2 wt % and 2.8 g L−1 d−1, respectively, and the PHB content and productivity of 68.5 wt% and 1.06 g L−1 h−1, which was slightly reduced as compared to the processes where production of ectoines and the biopolymer were studied separately.