Fumaric acid production

Maleic anhydride and the two diacid isomers were first prepared in the 1830s (1) but commercial manufacture did not begin until a century later. In 1933 the National Aniline and Chemical Co., Inc., installed a process for maleic anhydride based on benzene oxidation using a vanadium oxide catalyst (2). Maleic acid was available commercially ia 1928 and fumaric acid production began in 1932 by acid-catalyzed isomerization of maleic acid. 

A large amount of rice bran caused excessive fungal growth rather than enhance fumaric acid production. In addition, we could produce fumaric acid without the addition of zinc and iron. Fungal culture broth containing approx 25 g/L of fumaric acid was directly employed for succinic acid conversion. The amount of glycerol and yeast extract required for succinic acid conversion was reduced to 70 and 30%, respectively, compared with the amounts cited in previous studies. 

In the present study, we evaluated a two-step process for succinic acid production. The first process was fumaric acid production by Rhizopus sp. using rice bran, and the second process was succinic acid production by Enterococcus faecalis RKY1 (5-7) using fungal culture broth obtained in the first process. We investigated the effects of rice bran on fumaric acid production and optimized the culture medium for fumaric acid fermentation. Furthermore, we optimized the culture conditions for succinic acid conversion from fumaric acid produced by the first process. 

To optimize the culture medium for fumaric acid production, we investigated the effects of rice bran concentrations and various carbon sources. When rice bran was used as a nitrogen source, the effects of additional elements (phosphate, magnesium, zinc, and iron) on fumaric acid production were also investigated. The medium previously reported by Zhou et al. (11) was used as the basal medium. Fermentations were performed in 250-mL Erlenmeyer flasks containing 100 mL of medium. 

Fig. 1. Effect of rice bran concentration on fumaric acid production. The culture conditions were as follows 250-mL flask containing 100 mL of medium at 35°C, 200 rpm for 4 d 50 g/L of initial glucose 15 g/L of CaC03 0.6 g/L of KH2P04 0.5 g/L of MgS04-7H20 0.0179 g/L of ZnS04-7H20 0.498 mg/L of FeS04-7H20.

Effect of Phosphate, Magnesium, Zinc, and Iron on Fumaric Acid Production Using Rice Bran ... 

With the use of rice bran as a nitrogen source, we studied the effect of phosphate, magnesium, zinc, and iron on fumaric acid production. Although magnesium, zinc, and iron did not cause any effect (Mg2+ trials 1 and 4, Zn2+ trials 1 and 3, Fe2+ trials 1 and 2), phosphate was crucial to fumaric acid production (trials 12 and 16). 

Batch fermentations were carried out using rice bran and glucose as nitrogen and carbon sources, respectively. Figure 4 shows the profile of fumaric acid production in a 2.5-L jar fermentor. The fumaric acid concentration reached 25.3 g/L. The yield (fumaric acid produced/glucose consumed) and the productivity were 52% and 0.21 g/(L-h), respectively. For the following experiment, the fungal culture broth was used as a medium for the bioconversion of fumaric acid into succinic acid. 

Zhou, Y., Du, J., and Tsao, G.T. 2002. Comparison of fumaric acid production by Rhizopus oryzae using different neutralizing agents. Bioprocess and Biosystems Engineering 25 179-181. 

Fumaric Acid Production with a Rotary Biofilm Contactor RBC. . 267... 

Fumaric acid production using immobilized Rhizopus cells has also been studied. Petruccioli et al. [74] immobilized R. arrhizus NRRL 1526 on polyurethane sponge to carry out repeated batch fumaric acid production from glucose syrup with KOH/KCO3 as the neutralization agent and CO2 source. Although the yield (12.3 g/1) is low, it provides the possibility of using immobilized Rhizopus for the continuous production of fumaric acid. 

Fumaric acid production from starch hydrolysate by R. arrhizus NRRL 1526 was studied by Federici et al. [75] in a 3-1 stirred-tank fermentor with CaCO, and KOH/KCO3 as the neutralizing agent and CO2 source. The fermentation conditions for fumaric acid production by this fungus from potato flour has been optimized by Moresi et al. [76]. 

Toward Engineering Rhizopus for Fumaric Acid Production... 

According to Rhodes et al. (1959) the accumulation of fumaric acid maybe attributed to low activity or absence of fumarase. However, a high in vitro activity of fumarase was measured during the fumaric acid production stage (Osmani and Scrutton, 1985 Kenealy et al., 1986). 

Direct proof that this reductive TCA pathway participates in fumaric acid production came from C NMR experiments using [1- C] and [U- C] glucose as a carbon source for R. oryzae (Kenealy et al., 1986). The unexpectedly high fumaric acid molar yields can thus be explained in terms of pyruvate carboxylation as the initial reaction of a reductive TCA pathway (Figure 15.1). 

Evidence for the carbon dioxide fixation mechanism for fumaric acid production came from two sources. Osmani and Scrutton (1985) discovered that R. oryzae... 

The operation of the cytosolic reductive TCA pathway for fumaric acid production in R. oryzae raises the question as to the unique role of the cytosolic fumarase in this fungus. 

It should be mentioned that with another Rhizopus strain. Ding et al. (2011) showed in cell extracts, in accordance to previous findings that lowering the urea concentrations in the medium from 2.0 to 0.1 g/L caused an increase of 300% in the cytosolic fumarase activity, accompanied with an increase in fumaric acid production. 

Thus, the cytosolic FUMR may be an important step for the accumulation of high concentrations of fumaric acid in R. oryzae. This conclusion can be better confirmed if fumaric acid production will increase as a result of the overexpression of FUMR in R. oryzae (Song et al., 2011 Zhang and Yang, 2012). 

TOWARD ENGINEERING RHIZOPUS FOR FUMARIC ACID PRODUCTION... 

Few platform organisms were suggested for fumaric acid production in addition to Rhizopus strains. E. coli has been chosen as a workhouse for the production of fumaric acid and many valuable chemicals including other organic... 

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