Malic acid production from the biodiesel coproduct crude glycerol by Aspergillusniger ATCC 9142, ATCC 10577 and ATCC 12846 was observed to occur with the highest malic acid level acid being produced by A. niger ATCC 12846. Fungal biomass production from crude glycerol was similar, but ATCC 10577 produced the highest biomass. Fungal biotransformation of crude glycerol into the commercially valuable organic acid malic acid appeared feasible.
The organic acid malic acid is a specialty chemical that has applications in metal cleaning, pharmaceuticals, plastics and foods as well as beverages . Malic acid is currently produced by chemical synthesis with the world production being about 40,000 tons annually . Malic acid can be produced by species of the fungus Aspergillus when grown on fermentable substrates [1–5]. In the fungus Penicillium sclerotiorum, an isolate was found to produce high calcium malate levels on a medium containing glucose as a carbon source and ammonium nitrate or corn steep liquor as a carbon source [6, 7].
An important problem for the biodiesel production industry is the utilization of processing coproducts. During biodiesel production, a coproduct stream containing glycerol, fatty acids and methylesters of fatty acids results, and it represents 10% of the coproduct formation . It is expected that large volumes of crude glycerol will be available for fermentation considering that more than 30 million gallons of biodiesel are being produced annually. It will be important to learn whether the low-value coproduct crude glycerol can undergo biotransformation to a more commercially valuable chemical such as malic acid.
In this study, three known malic acid-producing strains of Aspergillus niger were screened for their ability to catalyze the biotransformation of crude glycerol into malic acid after 192 h of growth at 25 °C. The production of cellular biomass by these strains was also examined in relation to the level of malic acid synthesized.
The known malic acid-producing strains A. niger ATCC 9142, A. niger ATCC 10577 and A. niger ATCC 12846 were used in this study  and obtained from the American Type Culture Collection, Manassas, VA, USA. Each strain of Aspergillus was inoculated into 10 mL potato dextrose broth (Difco Laboratories, Inc., Detroit, MI, USA) using a loopful of fungal mycelium, and the culture was grown for 48 h at 25 °C. The growth medium contained 5.51 mM potassium phosphate monobasic (Fisher Scientific Co., Fair Lawn, NJ, USA), 4.31 mM potassium phosphate dibasic (Fisher Scientific Co., Fair Lawn, NJ, USA), 0.41 mM magnesium sulfate heptahydrate (Mallinckrodt Baker, Inc., Paris, KY, USA), 0.68 mM calcium chloride dihydrate (J. T. Baker Chemical Co., Phillipsburg, NJ, USA), 0.09 mM sodium chloride (Sigma Chemical Co., St. Louis, MO, USA), 0.02 mM ferrous sulfate heptahydrate (Sigma Chemical Co., St. Louis, MO, USA), 30.27 mM ammonium sulfate (Mallinckrodt Baker, Inc., Paris, KY, USA), 651.21 mM potassium carbonate (Sigma Chemical Co., St. Louis, MO, USA) and 10% crude glycerol. The potassium carbonate was added as a neutralizing agent , and its pH was adjusted to 6.0. The crude glycerol, obtained from a local soy biodiesel producer, contained 79% glycerol and 16% methyl esters of fatty acids, which was determined enzymatically and by chemical analysis . The fungal inoculum (17%) was added to 10 mL of sterilized medium (pH 6.0) in a sterile 125 mL Erlenmeyer flask and grown for a period of 192 h at 25 °C (200 rpm). After 192 h, each of the three liquid cultures was processed using the following procedure. Each culture was filtered through a Whatman No. 1 filter (Whatman International Ltd., Maidstone, UK), and the fungal mycelium in each culture was collected. The resultant supernatant for each culture was collected for subsequent malic acid determination, while the wet fungal mycelium was saved for biomass determinations.
Malic acid production by the Aspergillus species was determined spectrophotometrically using a malate dehydrogenase assay . The supernatant was assayed for its malic acid content using a spectrophotometric assay. The assay mix (1 mL) contained 798 mM glycine buffer pH 9.0, 13.3 mM disodium EDTA dihydrate, 2 mM 3-acetylpyridine-adenine dinucleotide and 42 units malate dehydrogenase and sample. Malic acid standards were also run using the assay. All assay reagents and the malic acid used to prepare the standards were obtained from the Sigma Chemical Co. (St. Louis, MO, USA). The reaction was monitored at 365 nm by following the increase in absorbance that is proportional to the concentration of malic acid present in the sample. The wet fungal biomass after 192 h was put in a preweighed beaker, weighed and dried to constant weight at 80 °C . All values represent the mean of three independent determinations involving three separate cultures. The Student’s t test was used during statistical analysis.
Although all the Aspergillus strains investigated were capable of producing malic acid from crude glycerol, ATCC 12486 produced the highest malic acid level after 192 h of fermentation in batch cultures from 10% crude glycerol (Figure 1). The difference in malic acid production between ATCC 12846 and ATCC 9142 was statistically significant (p<0.01). As can be observed in Figure 1, ATCC 10577 also produced a higher malic acid level after 192 h on 10% crude glycerol than did ATCC 9142 with the difference in malic acid production being statistically significant (p<0.05). Prior investigations have studied malic acid production by Aspergillusflavus ATCC 13697. ATCC 13697 produced 36.4 g/L malic acid from 10% glucose as the carbon source and calcium carbonate as a neutralizing agent when grown for 192 h at 25 °C . Malic acid production by ATCC 13697 can be inhibited in the presence of the protein synthesis inhibitor cycloheximide because synthesis of the major isoenzyme of malate dehydrogenase is blocked [2, 3]. Malic acid was synthesized in ATCC 13697 from pyruvate with oxaloacetate serving as an intermediate due to the enzyme pyruvate decarboxylase [2, 10]. It was determined that A. flavus ATCC 13697 as well as A. niger ATCC 9029, ATCC 9142 and ATCC 10577 were capable of malic acid production after 135 h at 30 °C in a 10% glucose-containing production medium . It was also shown that A. niger ATCC 9142 and ATCC 10577 had a higher NAD-dependent malate dehydrogenase activity in its cytosolic and mitochondrial fractions than did A. flavus ATCC 13697 or A. niger ATCC 9029 when the strains were grown on the glucose-containing medium . The higher dehydrogenase activity in ATCC 9142 or ATCC 10577 may be responsible for their increased malic acid production on glucose. The concentration of malic acid produced by A. niger ATCC 9142 or ATCC 10577 on thin stillage was about 1.6-fold higher than what was observed for A. flavus ATCC 13697 . Only A. niger ATCC 9029 produced a lower malic acid level than did ATCC 13697 . It was suggested that ATCC 9142 or ATCC 10577 could utilize the glycerol present in the thin stillage more effectively than the other strains to produce a higher malic acid level . This would seem to be confirmed in the present study since all three A. niger strains could effectively utilize the glycerol in the crude glycerol to produce malic acid.
Biomass production by the Aspergillus strains following growth on the crude glycerol for 192 h at 25 °C was next investigated. As can be seen in Figure 1, the cell dry weights for ATCC 9142, ATCC 10577 and ATCC 12846 were capable of producing cellular biomass from the 10% crude glycerol-containing medium after 192 h. The highest biomass level was produced by A. niger ATCC 10577 from the 10% crude glycerol (Figure 1). Aspergillus niger ATCC 9142 and ATCC 12846 produced much lower biomass levels than did ATCC 10577 (Figure 1), but the difference in biomass production between A. niger ATCC 9142 and ATCC 12846 was statistically significant (p<0.01). Similar to this study, biomass production by A. niger ATCC 10577 grown on thin stillage was higher than biomass production by A. niger ATCC 9142 or A. flavus ATCC 13697 after 192 h at 25 °C .
Overall, it was concluded that the three A. niger strains could utilize 10% crude glycerol to produce malic acid and biomass after 192 h at 25 °C, but the acid and biomass levels produced were strain dependent. It appeared that crude glycerol could be used as an effective substrate for fungal malic acid production. To bring this project to fruition, the optimization of fungal malic acid production from crude glycerol will be necessary. In addition, it will be necessary to purify malic acid from the other organic acids produced by A. niger such as succinic acid or fumaric acid. The difficulty in separating the individual organic acids has previously been reported  and will require novel separation techniques to be developed.
Financial support of this work was provided by the South Dakota Agricultural Experiment Station Grant SD00H434-12. The technical assistance of Jessica L. Peterson was greatly appreciated.
1. Goldberg I, Rokem JS, Pines O. Organic acids: old metabolites, new themes. J Chem Technol Biotechnol 2006;81:1601–11. Search in Google Scholar
2. Peleg Y, Stieglitz B, Goldberg I. Malic acid accumulation by Aspergillus flavus. I. Biochemical aspects of acid biosynthesis. Appl Microbiol Biotechnol 1988;28:69–75. Search in Google Scholar
3. Peleg Y, Barak A, Scrutton MC, Goldberg I. Malic acid accumulation by Aspergillus flavus. III. 13C NMR and isoenzymes analyses. Appl Microbiol Biotechnol 1989;30:176–83. Search in Google Scholar
4. Battat E, Peleg Y, Bercovitz A, Rokem JS, Goldberg I. Optimization of L-malic acid production by Aspergillus flavus in a stirred fermentor. Biotechnol Bioeng 1991;37:1108–16. Search in Google Scholar
5. West TP. Malic acid production from thin stillage by Aspergillus species. Biotechnol Lett 2011;33:2463–7. Search in Google Scholar
6. Wang Z-P, Wang G-Y, Khan I, Chi Z-M. High-level production of calcium malate from glucose by Penicillium sclerotiorum K302. Bioresour Technol 2013;143:674–7. Search in Google Scholar
7. Khan I, Nazir K, Wang Z-P, Liu G-L, Chi Z-M. Calcium malate overproduction by Penicillium viticola 152 using the medium containing corn steep liquor. Appl Microbiol Biotechnol 2014;98:1539–46. Search in Google Scholar
8. Tapasvi D, Wiesenborn D, Gustafson C. Process model for biodiesel production from various feedstocks. Trans ASAE 2005;48:2215–21. Search in Google Scholar
9. West TP. Citric acid production by Candida species grown on a soy-based crude glycerol. Prep Biochem Biotechnol 2013;43:601–11. Search in Google Scholar
10. Bercovitz A, Peleg Y, Battat E, Rokem JS, Goldberg I. Localization of pyruvate carboxylase in organic acid-producing Aspergillus strains. Appl Environ Microbiol 1990;56:1594–7. Search in Google Scholar
11. Kertes AS, King CJ. Extraction chemistry of fermentation carboxylic acids. Biotechnol Bioeng 1985;28:269–82. Search in Google Scholar
©2015 by De Gruyter