Alginate production by mutants of Azotobacter vinelandii: advanced strategies for cultivation under microaerophilic conditions and overpressure

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Highly viscous fermentation broths are one of the problems frequently encountered in the development and scaling of bioprocesses. The production of polysaccharides can be considered as an example of such processes. In the field of microbial polysaccharides, alginates are counted among the most important ones. Alginate is a polymer composed of beta-D-mannuronic acid (M) and alpha-L-guluronic (G) acid and produced by brown algae and bacterial species such as Azotobacter vinelandii and Pseudomonas aeruginosa. Alginates have a wide scope of application due to their use as viscosifiers and gel forming agents, making them of great interest to several industrial areas. Because most details of the alginate biosynthesis are known, as well as the metabolic relationships of this pathway with other pathways present in A. vinelandii, it was possible to generate overproducing strains of this biopolymer. Moreover, genetic modifications allow to influence the physical and chemical properties of the produced alginate. In addition, previous studies have shown that for very low dissolved oxygen tensions (microaerophilic conditions), it is possible to manipulate both the molecular mass as well as the degree of acetylation of alginate produced by A. vinelandii. For this reason the highest priority of this proposal is to investigate the potential of microaerophilic conditions to significantly maximize the alginate formation with specific chemical characteristics by genetically modified strains of A. vinelandii. The formation of alginate leads to a non-Newtonian fluid behavior of the fermentation broth with a significant impact on mixing homogeneity, power input into the liquid, heat removal and in general on the reproduction of results. Thus, the scale up from shake flask to stirred tank reactor is a major challenge for this biological system. Process scale up and reproduction of results on shake flask level, lab scale stirred tank reactor as well as in a 50 L pilot scale pressure reactor will therefore also be an important part of this proposal. In addition, this project will give students and scientists the opportunity for research stays in the Mexican group of Dr. Peña at UNAM Institute of Biotechnology and the German group of Dr. Regestein at RWTH Aachen University.