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Calcium carbonate has been extensively used as a neutralising agent in acid-forming microbial processes. The effect of increasing calcium carbonate concentrations on Rhizopus delemar has not been previously investigated. In this study, an evaluation of fumaric acid (FA) and malic acid (MA) production was conducted at three CaCO3 concentrations in shake flask cultivations. Increased CaCO3 concentrations resulted in the co-production of FA and MA in the first 55 h of the fermentation (regime 1), and the subsequent depletion of FA thereafter (regime 2). Three factors were highlighted as likely causes of this response: insoluble solids, metal ion concentrations, and pH. Further shake flask cultivations as well as a continuous fermentation with immobilised R. delemar were used to explore the effect of the three factors on regime 1 and 2. Insoluble solids were found to have no effect on the response in either regime 1 or 2. Increasing the aqueous calcium ion concentrations to 10 g L−1 resulted in a three-fold increase in MA titres (regime 1). Moreover, an increase in pH above 7 was associated with a drop in FA concentrations in regime 2. Further tests established that this was due to the hydration of FA to MA, influenced by high pH conditions (7 or higher), nitrogen starvation, and glucose depletion. Anaerobic conditions were also found to significantly improve the hydration process. This study presents the first investigation in which the production of FA followed by in situ hydration of FA to MA with R. delemar has been achieved.

The Effect of pH, Metal Ions, and Insoluble Solids on the Production of Fumarate and Malate by Rhizopus delemar in the Presence of CaCO3