Investigation of growth modes of the lithoautotrophic bacterium Cupriavidus necator

Abstract: The microorganism Cupriavidus necator is a very interesting bacterium that has been established as a model microorganism for its capability to produce polyhydroxyalkanoates, more specifically polyhydroxybutyrate (PHB), a potential precursor/raw material for bioplastics. Lately, the scientific community has raised interest in the chemolithoautotrophic nature of C. necator as it can be grown using CO2 and H2 as its only carbon and energy sources, respectively. Alternatively, it can also be grown under organoautotrophic conditions, where formic acid is used as substrate that is further metabolized in the cell to obtain CO2 and reduced NADPH. The organoautotrophic growth mode is presumably similar to lithoautotrophic growth, as both modes only differ in the initial step of substrate assimilation. An initial attempt to characterize growth under organoautotrophic conditions was performed and it was observed that the biomass yield was almost three times lower than using fructose as substrate for heterotrophic mode. Furthermore, growth under anaerobic conditions was explored as an alternative to avoid the potential explosion risk from mixtures of hydrogen and oxygen. It was found that respiration, either using oxygen or nitrate as electron acceptor, is needed for ATP production in C. necator and thus to support growth. To facilitate growth and reoxidation of NADH also under anaerobic conditions a new strain was generated in which the ethanol fermentation pathway was introduced. A preliminary characterization of the fermentative capacity of this strain showed that no ethanol was produced when grown organoautotrophically.