Abstract: Even though microorganisms can often grow in defined media, they are frequently cultured in rich media containing complex components like yeast extract. A drawback of using rich media is the effect of secondary substrates from complex components on the metabolism, which can change the anabolism and the formation of products, complicating the interpretation of experimental results. Rich media containing yeast extract is generally used to grow Clostridium pasteurianum. In this work, we describe the development of a minimal medium for C. pasteurianum combining rational media design, transfers in batch bottles and continuous bioreactors experiments. The media were designed based on literature, the elements needed in the metabolism, and a general chemical formula for the composition of biomass. The media were tested by cultivations in chemostat and batch bottles. Microbial growth was not sustained in an inorganic medium with glucose over batch bottles transfers. In contrast, a medium with glucose supplemented with para-aminobenzoic acid (PABA), biotin and cysteine, supported growth in chemostat (12 retention times) and in batch bottles transfers. Growth was also maintained in the same medium without cysteine during the 12 retention times of chemostat operation, but at a lower dilution rate, showing that cysteine enhanced the growth rate of C. pasteurianum despite not being essential. Microbial growth was sustained through batch bottle transfers in media with PABA only and with biotin only, apart from glucose and cysteine. Therefore, it was concluded that PABA and biotin are essential for the growth of C. pasteurianum without yeast extract, only one—any—amongst both being needed. Key points: • Clostridium pasteurianum is auxotrophic on B vitamins. • Both biotin and PABA suffice to support growth in an otherwise inorganic medium. • Cysteine was not essential but increased its growth rate.