Background: The four-carbon dicarboxylic acids of the tricarboxylic acid cycle (malate, fumarate and succinate) remain promising bio-based alternatives to various precursor chemicals derived from fossil-based feed stocks. The double carbon bond in fumarate, in addition to the two terminal carboxylic groups, opens up an array of downstream reaction possibilities, where replacement options for petrochemical derived maleic anhydride are worth mentioning. To date the most promising organism for producing fumarate is Rhizopus oryzae (ATCC 20344, also referred to as Rhizopus delemar) that naturally excretes fumarate under nitrogen-limited conditions. Fumarate excretion in R. oryzae is always associated with the co-excretion of ethanol, an unwanted metabolic product from the fermentation. Attempts to eliminate ethanol production classically focus on enhanced oxygen availability within the mycelium matrix. In this study our immobilised R. oryzae process was employed to investigate and utilise the Crabtree characteristics of the organism in order to establish the limits of ethanol by-product formation under growth and non-growth conditions. Results: All fermentations were performed with either nitrogen excess (growth phase) or nitrogen limitation (production phase) where medium replacements were done between the growth and the production phase. Initial experiments employed excess glucose for both growth and production, while the oxygen partial pressure was varied between a dissolved oxygen of 18.4% and 85%. Ethanol was formed during both growth and production phases and the oxygen partial pressure had zero influence on the response. Results clearly indicated that possible anaerobic zones within the mycelium were not responsible for ethanol formation, hinting that ethanol is formed under fully aerobic conditions as a metabolic overflow product. For Crabtree-positive organisms like Saccharomyces cerevisiae ethanol overflow is manipulated by controlling the glucose input to the fermentation. The same strategy was employed for R. oryzae for both growth and production fermentations. It was shown that all ethanol can be eliminated during growth for a glucose addition rate of 0.07 g L −1 h −1. The production phase behaved in a similar manner, where glucose addition of 0.197 g L −1 h −1 resulted in fumarate production of 0.150 g L −1 h −1 and a yield of 0.802 g g −1 fumarate on glucose. Further investigation into the effect of glucose addition revealed that ethanol overflow commences at a glucose addition rate of 0.395 g g −1 h −1 on biomass, while the maximum glucose uptake rate was established to be between 0.426 and 0.533 g g −1 h −1. Conclusions: The results conclusively prove that R. oryzae is a Crabtree-positive organism and that the characteristic can be utilised to completely discard ethanol by-product formation. A state referred to as "homofumarate production" was illustrated, where all carbon input exits the cell as either fumarate or respiratory CO 2. The highest biomass-based

Fumaric acid is widely used in the food and beverage, pharmaceutical and polyester resin industries. Rhizopus oryzae is the most successful microorganism at excreting fumaric acid compared to all known natural and genetically modified organisms. It has previously been discovered that careful control of the glucose feed rate can eliminate the by-product formation of ethanol. Two key parameters affecting fumaric acid excretion were identified, namely the medium pH and the urea feed rate. A continuous fermentation with immobilised R. oryzae was utilised to determine the effect of these parameters. It was found that the selectivity for fumaric acid production increased at high glucose consumption rates for a pH of 4, different from the trend for pH 5 and 6, achieving a yield of 0.93 gg−1. This yield is higher than previously reported in the literature. Varying the urea feed rate to 0.255 mgL−1h−1 improved the yield of fumaric acid but experienced a lower glucose uptake rate compared to higher urea feed rates. An optimum region has been found for fumaric acid production at pH 4, a urea feed rate of 0.625 mgL−1h−1 and a glucose feed rate of 0.329 gL−1h−1.

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.

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The hydrolysis of lignocellulosic biomass opens an array of bioconversion possibilities for producing fuels and chemicals. Microbial fermentation is particularly suited to the conversion of sugar-rich hydrolysates into biochemicals. Rhizopus oryzae ATCC 20344 was employed to produce fumaric acid from glucose, xylose, and a synthetic lignocellulosic hydrolysate (glucose–xylose mixture) in batch and continuous fermentations. A novel immobilised biomass reactor was used to investigate the co-fermentation of xylose and glucose. Ideal medium conditions and a substrate feed strategy were then employed to optimise the production of fumaric acid. The batch fermentation of the synthetic hydrolysate at optimal conditions (urea feed rate 0.625mgL−1h−1 and pH 4) produced a fumaric acid yield of 0.439gg−1. A specific substrate feed rate (0.164gL−1h−1) that negated ethanol production and selected for fumaric acid was determined. Using this feed rate in a continuous fermentation, a fumaric acid yield of 0.735gg−1 was achieved; this was a 67.4% improvement. A metabolic analysis helped to determine a continuous synthetic lignocellulosic hydrolysate feed rate that selected for fumaric acid production while achieving the co-fermentation of glucose and xylose, thus avoiding the undesirable carbon catabolite repression. This work demonstrates the viability of fumaric acid production from lignocellulosic hydrolysate; the process developments discovered will pave the way for an industrially viable process.

It has been proposed that the microbial bioproduction of chemical precursors could reduce societal overreliance on the petrochemical industry. However, economic implementation remains elusive due to a lack of understanding of the mechanisms involved. The current study evaluated malic acid production using a novel immobilized Aspergillus oryzae fungal bed bioreactor. The experimental procedures involved two different nitrogen sources (urea and (NH4)2SO4), two‐phase growth/production and simultaneous single‐step growth/production runs, the presence and absence of added CO2, the presence and absence of pH control, and the use of glucose and dl‐malate as the carbon sources. The experimental malic acid titers were remarkably poor (between 1.7 and 3.3 g.L−1) in comparison with CaCO3 buffered malic acid titers from literature (concentrations in excess of 100 g.L−1 have been reported reported). In contrast, citric acid was observed to be the main metabolite. The dl‐malate carbon source was consumed with concomitant growth, indicating A. oryzae′s ability to utilize both d and l enantiomers of malic acid. Detailed metabolic flux analyses predicted partial consumption of urea during growth, while (NH4)2SO4 was fully utilized. During the urea runs, significant amounts of glycogen were accumulated in the biomass – which were utilized as substrate during the production run. Nitrogen limitations induced significant lipid production, corresponding to citric acid accumulation in the media; the mechanism for fungal lipid biosynthesis involves citric acid excretion at the expense of malic acid. These results indicate a possible calcareous trigger for malic acid production as opposed to the traditionally accepted nitrogen starvation mechanism – nitrogen starvation resulted in lipid accumulation with corresponding malate loss. © 2022 The Authors. Biofuels, Bioproducts and Biorefining published by Society of Industrial Chemistry and John Wiley & Sons Ltd.