Z. Naturforsch. 2015; aop
Research note Thomas P. West*
Fungal biotransformation of crude glycerol into malic acid DOI 10.1515/znc-2015-0115 Received April 24, 2015; revised May 29, 2015; accepted May 30, 2015
Abstract: Malic acid production from the biodiesel coproduct crude glycerol by Aspergillus niger 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. Keywords: biomass; crude glycerol; fungus; malic acid.
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.
2 Materials and methods 2.1 Fungal strains and culture conditions
1 Introduction The organic acid malic acid is a specialty chemical that has applications in metal cleaning, pharmaceuticals, plastics and foods as well as beverages [1]. Malic acid is currently produced by chemical synthesis with the world production being about 40,000 tons annually [1]. 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 [8]. It is expected that large volumes of crude glycerol will be
*Corresponding author: Thomas P. West, Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57007, USA, Phone: +605-688-5469, Fax: +605-688-6364, E-mail: [email protected]
The known malic acid-producing strains A. niger ATCC 9142, A. niger ATCC 10577 and A. niger ATCC 12846 were used in this study [5] 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 [5], 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 [9]. 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
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2 West: Fungal malic acid biotransformation
acid determination, while the wet fungal mycelium was saved for biomass determinations.
2.2 Malic acid and biomass determinations Malic acid production by the Aspergillus species was determined spectrophotometrically using a malate dehydrogenase assay [5]. 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 [5]. All values represent the mean of three independent determinations involving three separate cultures. The Student’s t test was used during statistical analysis.
3 Results and discussion 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
Research note Thomas P. West*
Fungal biotransformation of crude glycerol into malic acid DOI 10.1515/znc-2015-0115 Received April 24, 2015; revised May 29, 2015; accepted May 30, 2015
Abstract: Malic acid production from the biodiesel coproduct crude glycerol by Aspergillus niger 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. Keywords: biomass; crude glycerol; fungus; malic acid.
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.
2 Materials and methods 2.1 Fungal strains and culture conditions
1 Introduction The organic acid malic acid is a specialty chemical that has applications in metal cleaning, pharmaceuticals, plastics and foods as well as beverages [1]. Malic acid is currently produced by chemical synthesis with the world production being about 40,000 tons annually [1]. 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 [8]. It is expected that large volumes of crude glycerol will be
*Corresponding author: Thomas P. West, Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57007, USA, Phone: +605-688-5469, Fax: +605-688-6364, E-mail: [email protected]
The known malic acid-producing strains A. niger ATCC 9142, A. niger ATCC 10577 and A. niger ATCC 12846 were used in this study [5] 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 [5], 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 [9]. 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
Brought to you by | Harvard University Authenticated Download Date | 7/28/15 3:35 PM
2 West: Fungal malic acid biotransformation
acid determination, while the wet fungal mycelium was saved for biomass determinations.
2.2 Malic acid and biomass determinations Malic acid production by the Aspergillus species was determined spectrophotometrically using a malate dehydrogenase assay [5]. 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 [5]. All values represent the mean of three independent determinations involving three separate cultures. The Student’s t test was used during statistical analysis.
3 Results and discussion 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
