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 "homofumarate production": rate of 0.243 g g - 1 h - 1 achieved a yield of 0.802 g g - 1 on glucose, indicating the bounds for developing an ethanol free process. The control strategy employed in this study in conjunction with the uncomplicated scalability of the immobilised process provides new direction for further developing bio-fumarate production.

中文翻译：

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用米根霉生产富马酸盐：利用 Crabtree 效应最大限度地减少乙醇副产物的形成。

背景 三羧酸循环中的四碳二羧酸（苹果酸、富马酸和琥珀酸）仍然是有前景的生物基替代品，可替代源自化石原料的各种前体化学品。除了两个末端羧基外，富马酸盐中的双碳键还开辟了一系列下游反应的可能性，其中值得一提的是石化衍生的马来酸酐的替代选择。迄今为止，最有希望生产富马酸盐的生物是米根霉（ATCC 20344，也称为 Rhizopus delemar），它在氮限制条件下自然分泌富马酸盐。米曲霉中的富马酸盐排泄总是与乙醇的共同排泄有关，乙醇是发酵中不需要的代谢产物。消除乙醇生产的尝试经典地集中在菌丝体基质内增强氧的可用性。在这项研究中，我们采用固定化米根霉工艺来研究和利用生物体的 Crabtree 特性，以确定生长和非生长条件下乙醇副产物形成的限制。结果所有发酵均在氮过量（生长期）或氮限制（生产阶段）的情况下进行，其中在生长和生产阶段之间进行培养基更换。最初的实验使用过量的葡萄糖进行生长和生产，而氧分压在 18.4% 和 85% 的溶解氧之间变化。在生长和生产阶段都形成了乙醇，并且氧分压对响应的影响为零。结果清楚地表明菌丝体内可能的厌氧区不是乙醇形成的原因，这暗示乙醇是在完全有氧条件下作为代谢溢出产物形成的。对于像酿酒酵母这样的 Crabtree 阳性生物，通过控制发酵过程中的葡萄糖输入来控制乙醇溢出。相同的策略用于米根霉的生长和生产发酵。结果表明，对于 0.07 g L - 1 h - 1 的葡萄糖添加率，在生长过程中可以消除所有乙醇。生产阶段以类似的方式表现，其中添加 0.197 g L - 1 h - 1 的葡萄糖导致产生 0.150 g L - 1 h - 1 的富马酸盐和 0.802 gg - 1 富马酸盐的产量。对葡萄糖添加影响的进一步研究表明，乙醇溢出以 0.395 gg - 1 h - 1 的葡萄糖添加速率对生物质开始，而最大葡萄糖摄取速率确定在 0.426 和 0.533 gg - 1 h - 1 之间。结论结果最终证明米根霉是一种Crabtree阳性菌，并且可以利用该特性完全消除乙醇副产物的形成。说明了一种称为“高富马酸盐生产”的状态，其中所有碳输入以富马酸盐或呼吸性 CO 2 形式离开细胞。基于生物质的最高“高富马酸盐生产”：0.243 gg - 1 h - 1 的速率在葡萄糖上实现了 0.802 gg - 1 的产量，表明开发无乙醇工艺的界限。