Phase shifts in the stoichiometry of rifamycin B fermentation and correlation with the trends in the parameters measured online

Abstract

Antibiotic fermentation processes are raw material cost intensive and the profitability is greatly dependent on the product yield per unit substrate consumed. In order to reduce costs, industrial processes use organic nitrogen substrates (ONS) such as corn steep liquor and yeast extract. Thus, although the stoichiometric analysis is the first logical step in process development, it is often difficult to achieve due to the ill-defined nature of the medium. Here, we present a black-box stoichiometric model for rifamycin B production via Amycolatopsis mediterranei S699 fermentation in complex multi-substrate medium. The stoichiometric coefficients have been experimentally evaluated for nine different media compositions. The ONS was quantified in terms of the amino acid content that it provides. Note that the black box stoichiometric model is an overall result of the metabolic reactions that occur during growth. Hence, the observed stoichiometric coefficients are liable to change during the batch cycle. To capture the shifts in stoichiometry, we carried out the stoichiometric analysis over short intervals of 8-16 h in a batch cycle of 100-200 h. An error analysis shows that there are no systematic errors in the measurements and that there are no unaccounted products in the process. The growth stoichiometry shows a shift from one substrate combination to another during the batch cycle. The shifts were observed to correlate well with the shifts in the trends of pH and exit carbon dioxide profiles. To exemplify, the ammonia uptake and nitrate uptake phases were marked by a decreasing pH trend and an increasing pH trend, respectively. Further, we find the product yield per unit carbon substrate to be greatly dependent on the nature of the nitrogen substrate. The analysis presented here can be readily applied to other fermentation systems that employ multi-substrate complex media.