In Gluconobacter oxydans cultivations on glucose, CaCO3 is typically used as pH-buffer. This buffer, however, has disadvantages: suspended CaCO3 particles make the medium turbid, thereby, obstructing analysis of microbial growth via optical density and scattered light. Upon searching for alternative soluble pH-buffers, bacterial growth and productivity was inhibited most probably due to osmotic stress. Thus, this study investigates in detail the osmotic sensitivity of G. oxydans ATCC 621H and DSM 3504 using the Respiratory Activity MOnitoring System. The tested soluble pH-buffers and other salts attained osmolalities of 0.32-1.19 osmol kg(-1). This study shows that G. oxydans ATCC 621H and DSM 3504 respond quite sensitively to increased osmolality in comparison to other microbial strains of industrial interest. Osmolality values of >0.5 osmol kg(-1) should not be exceeded to avoid inhibition of growth and product formation. This osmolality threshold needs to be considered when working with soluble pH-buffers.