Malic Acid Production Using Wild Type Microorganisms

As an intermediate of the TCA cycle, malic acid can be accumulated as a fermentation end-product by various microorganisms including filamentous fungi, yeasts, and bacteria. However, only a few wild-type filamentous fungi such as Aspergillus and Penicillium have the native ability to produce malic acid in large quantities from glucose and other carbon sources.

Aspergillus species are well known strains for malic acid production. Aspergillus flavus was the first patented strain for malic acid production in 1963. Through process optimization, A. flavus achieved a maximal malic acid titer of 113g/L in 190h. However, it has never been applied for large-scale production of malic acid due to its production of carcinogenic aflatoxins during the fermentation process. Aspergillus oryzae is generally regarded as safe (GRAS). Given its high similarity with A. flavus, A. oryzae NRRL 3488 was investigated for the production of malic acid. A titer of 30.27 g/L malic acid was obtained with a yield of 0.98mol/mol under high glucose and nitrogen starvation conditions. A. oryzae DSM1863 was used to produce malic acid from the waste substrate glycerol and the renewable carbon source xylose as, reaching product titers of 39.40 and 45.43g/L, respectively. Aspergillus niger is a well-known industrial workhorse for the production of organic acids, and its application for malic acid production has received increasing attention in recent years. A. niger strains ATCC 9142 and ATCC 10577 were investigated for the production of malic acid from thin stillage, and achieved product titers of 17 and 19g/L, respectively. When using crude glycerol as feedstock, A. niger ATCC 12486 could produce 23g/L malic acid after 192h at 25°C.

Several Penicillium species such as P. viticola 152 and P. sclerotiorum K302 isolated from marine environments were reported to be good malic acid producers, respectively accumulating up to 131 and 71.67g/L L-malic acid from glucose in 10L fermenters. The titer of 131g/L with a yield of 1.34mol/mol glucose and a productivity of 1.36g/L/h represents the highest malic acid production achieved using Penicillium to date.

Yeasts such as S. cerevisiae are excellent platforms for the biological production of industrial chemicals and have also been investigated for fermentative malic acid production. Malic acid was detected as a by-product in the yeast fermentation process as early as 1924. Afterwards, at least eight S. cerevisiae strains were tested in flasks to produce malic acid, but only two strains could synthesize more than 1 g/L of malic acid in 7 days. Zygosaccharomyces rouxii is an osmotolerant yeast associated with foods of low water activity. Z. rouxii V19 isolated from high-sugar fermented foods was able to produce 74.90g/L of malic acid with a yield of 0.52mol/mol from 193g/L glucose within 15 days under optimized conditions.

Some mushrooms also produce useful materials such as organic acids, and Schizophyllum commune IFO-4928 was able to produce 18g/L of malic acid from glucose under optimized conditions. In addition, the yeast-like fungus Aureobasidium pullulans was identified as a proficient polymalic acid (PMA) producer, reaching a product titer of 47g/L from glucose. PMA is a linear anionic C4-polyester consisting of L-malic acid monomers. Recently, many researchers attempted to produce malic acid through the hydrolysis of PMA, and 144.2g/L L-malic acid was obtained following purification and hydrolysis of 123.7g/L PMA, which was produced by fed-batch fermentation using cells immobilized in a fibrous-bed bioreactor (FBB). This process provided a novel approach for malic acid production.