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

Abstract

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 10g/L 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.

Fumaric acid production from fermented oil palm empty fruit bunches using fungal isolate K20: a comparison between free and immobilized cells

“…Although the final FA concentration for fermentation with free and immobilized cells was slightly different (31.23 and 32.03 g/L, respectively), the volumetric productivity for fermentation with immobilized cells (1.335 g/L/h) was six times higher than that for fermentation with free cells (0.217 g/L/h). Ronoh et al (2022) reported the effect of CaCO3 on the co-production of FA and malic acid by the immobilized R. delemar . The increased concentration of calcium ions to 10 g/L resulted in a three-fold enhancement of MA titres.…”

“…Thus, fungal cell immobilization for fermentation is complex process influenced by various parameters. One crucial parameter is fungal morphology, which is directly affected by culture conditions, including agitation rate, temperature, medium composition, initial spore concentration, and the immobilization matrix used ( Ronoh et al, 2022 ). Our study demonstrated that, despite the potential use of immobilized cells for FA fermentation, they proved to be less efficient and productive compared to free cells.…”

Optimization of l-malic acid production from acetate with Aspergillus oryzae DSM 1863 using a pH-coupled feeding strategy

“…With 60 or 100 g/L CaCO 3 compared to 20 g/L, carbonate was available until the end of the fermentation, which prevented a drop in pH and resulted in a higher malic to fumaric acid ratio. The authors ruled out the presence of insoluble solids as influencing factor but found increased ion concentrations and different pH values to affect the production profile [ 39 ]. Schmitt et al also observed a positive effect of calcium carbonate on malic acid productivity and associated it with its ability to control the pH as buffering agent, although the authors pointed out that this may not be its only function [ 32 ].…”

“…Ronoh et al reported a decreased glucose consumption and fumaric acid production of R. delemar with 20 g/L Ca 2+ in the medium, which suggests inhibition. However, they also observed an increased, albeit delayed, formation of malic acid which might be attributed to a stimulatory effect of Ca 2+ on the enzyme fumarase [ 39 ]. The results described in all these references were obtained for cultivations using glucose as carbon source.…”

Malic acid production by Aspergillus oryzae: the immobilized fungal fermentation route

“…in which it was further proposed that many of the effects and interactions of Ca 2+ with fungal physiology link to the maintenance of Ca 2+ homeostasis, with multiple channels and transporters dedicated to this task. Consequently, the magnitude of these effects is likely to be complex with synchronous interactions likely -as was observed in a recent study by Ronoh et al 44 in which a compounded interaction between Ca 2+ and pH were observed to affect the production of MA by Rhizopus oryzae.…”