The effect of pH, metal ions, and insoluble solids on the production of fumarate and malate by Rhizopus delemar in the presence of CaCO3

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.SUPPLEMENTARY MATERIAL : FIGURE S1. Metabolite distribution of Rhizopus delemar with the minor components including biomass, succinic acid and pyruvic acid. Cultures were incubated at 34 °C, 200 rpm in the presence of 20 g/L CaCO3 . FIGURE S2. Metabolite distribution of Rhizopus delemar with the minor components including biomass, succinic acid and pyruvic acid. Cultures were incubated at 34 °C, 200 rpm in the presence of 60 g/L CaCO3 . FIGURE S3. Metabolite distribution of Rhizopus delemar with the minor components including biomass, succinic acid and pyruvic acid. Cultures were incubated at 34 °C, 200 rpm in the presence of 60 g/L CaCO3 . FIGURE S4. Extracellular concentrations of glycerol during shake ask cultivation of R. delemar with diferent plaster sand concentrations. Results are the mean of triplicate experiments and error bars indicate the standard deviation. FIGURE S5. Extracellular concentrations of glycerol during shake ask cultivation of R. delemar with different calcium ion concentrations. 20 g L−1 CaCO3 was used for pH control as a baseline amount in these experiments and additional 5, 10 and 20 g L−1 calcium ions were added onto the baseline in the form of CaCl3 .DATA AVAILABITY STATEMENT : The data presented in this study are openly available in the University of Pretoria Research Data Repository at DOI: 10.25403/UPresearchdata.19229979.https://www.mdpi.com/journal/catalystsdm2022Chemical Engineerin