Fig. 4
From: CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16
Chemolithoautotrophic fermentations of AppA production strains of C. necator H16 PHB-4. AppA expression from the RepPar plasmid was driven either by the membrane bound hydrogenase (Pmbh) or the chromosomal Calvin-Benson-Bassham (Pcbb_chr.) promoter. Cells were cultivated in optimized mineral media without any antibiotics using a lab-scale fermenter. (A) During fermentation of the Pmhb and the Pcbb_chr strains, oxygen partial pressure in the gas feed was adjusted to maintain 2% dissolved oxygen in the media. The consumption of ammonia as nitrogen source was monitored by daily sampling and an enzymatic assay. (B) Cell growth and the associated acidification of the culture media were observed by OD600 and pH measurements of daily samples taken for both strains, Pmbh and Pcbb_chr, during the fermentation process. (C) AppA production was determined by immunoblot with an His-tag specific antibody for each cultivation day. As loading control, the PonceauS staining of the corresponding membrane is shown. (D) AppA activities were evaluated by measuring the phosphate released from phytic acid (15 min, 37 °C), due to the addition of cell lysate from the Pmbh and the Pcbb_chr strain