antarctica NRRL Y-7808 was found to require the most base
addition in order to maintain the pH of the culture (data not
shown). Its culture supernatant showed the production of a substantial
amount of an unknown compound when analyzed by
HPLC. This strain was selected for further work. A second P.
antarctica strain (NRRL Y-8295) did not exhibit significant acid
production from glucose. Mass spectral analysis of the screening
culture extract of P. antarctica NRRL Y-7808 revealed the major
compound to be itaconic acid as identified by a mass spectral
library. The HPLC retention time of authentic itaconic acid was
the same as the culture product.
The initial screening medium was modified by dropping the
concentrations of FeSO4, ZnSO4, and MnSO4 to 0.005 mM for
all from 0.18, 0.07, and 0.12 mM, respectively. This new medium
was used as a base to test for the effects of magnesium, iron, calcium,
and nitrogen concentration on the yield of itaconic acid in
flask experiments. No effect on itaconic acid yield was seen at
magnesium concentrations of 10 and 20 mM or iron concentrations
between 0.01 and 1 mM, either with or without CaCl2 at
0.2 mM. All cultures, including those grown on the initial screening
medium, produced approximately 30 g/L itaconic acid from
80 g/L glucose in the starting medium (data not shown), a 37.5%
yield. This yield is similar to that seen by Tabuchi et al. for a
Candida strain grown under phosphate-limited conditions [7].
The ability of P. antarctica to utilize glycerol and sugars
other than glucose to produce IA was also tested (Fig. 1). In this
experiment, the carbon source concentration was 40 g/L and the
nitrogen concentration was adjusted to maintain the C/N ratio
used in the previous experiment. The monosaccharides glucose
and fructose were utilized most efficiently, followed by the disaccharides
sucrose and maltose. Lactose and glycerol were the
poorest substrates, although both did yield slight amounts of IA
(Fig. 1)