Bioresource Technology 65 (1998) 243-245
@ 1998 Elsevier Science Ltd. All rights reserved
ELSEVIER
PII:S0960-8524(98)00026-1
Short Communication
Improvement of Citric Acid Production
by Aspergillus niger with Addition of
Phytate to Beet Molasses
Abstract
Phytate is an important plant constituent and can be
found in the seeds of cereals and legumes. Phytic acid
has 12 replaceable protons in the phytic molecule,
giving it the ability to complex with multivalent cations.
In this study, the phytate was used as an additive to
enhance citric acid production by Aspergillus niger
from an untreated beet molasses. The addition of
phytate caused increase of reducing sugar utilization
and enhanced citric acid accumulation; however, the
mycelial growth was slightly inhibited. The effect of
phytate addition was found to be dependent on
concentration and the stage of fermentation at which
phytate was added. When added at the beginning of
incubation, the optimal concentration of phytate in the
medium for citric acid production was lOg~l, which
resulted in about a &l-fold increase in citric acid
accumulation. During the fermentation, addition of
16 g/l phytate to the medium, after 3 days incubation,
gave the maximal citric acid production which was
about 2.4-fold higher than the control experiment.
@ 1998 Elsevier Science Ltd. All rights reserved
Key words: Citric acid, fermentation, Aspergillus
niger, phytate.
INTRODUCTION
Citric acid, an intermediate of the tricarboxylic acid
cycle, is produced almost exclusively by fermentation
with Aspergillus niger, and widely used in the food,
beverage, chemical, pharmaceutical and other indus-
tries (Wang & Liu, 1996). Considerable amounts of
citric acid are required by large-scale industrial
processes. In view of the numerous applications of
citric acid, its production by fermentation is
increasing continually. About 400000 tons of citric
acid are produced annually by fermentation.
The most widely used industrial substrate for
citric acid production by Aspergillus niger is beet
molasses, a by-product of sugar production. In
243
Printed in Great Britain
0960-8524/98 $19.00
recent years, however, the increasing recovery
capacity for sugar-refining processes has lowered the
quantity and quality of sugars in the molasses, so
decreasing their fermentability.
Citric acid production by Aspergillus niger is influ-
enced by a number of culture parameters, especially
trace-metal ions. The presence of trace metals in
toxic concentrations can be a significant problem
during the fermentation of crude substrates into
useful chemical products. Concentrations of trace
metals in culture media may be controlled in either
of two ways. One method is to purify the medium so
as to remove certain metal ions and then add known
amounts of the required metal salts. The second
method is to add metal-chelating agents to the
medium to decrease the concentration of free metal
ions to the desired values. This method has the
advantage that the metal complex acts as a 'metal
buffer', which reversibly dissociates to release ions as
they are utilized by growing organisms or combines
with excess metal ions added to system.
The removal of iron and other biologically
important metals from molasses by the formation of
complexes with ferrocyanide ion has been exten-
sively used for citric acid production by Aspergillus
niger (Clark et al., 1965; Choudhary & Pirt, 1966).
The addition of potassium ferrocyanide enhanced
the yield of citric acid. The effects of complexing
agents on the sensitivity to metals of ethanol
production from sugar cane molasses by the use of
Saccharomyces cerevisiae have been investigated
(Oderinde et al., 1986; Oderinde et al., 1990). Ergun
et al. (1997) studied the influence of ethylenediami-
netetraacetic acid (EDTA), potassium ferrocyanide
and zeolite X on ethanol production from sugar beet
molasses by S. cerevisiae.
Phytic acid (myoinositol hexaphosphate) or its
calcium salt, phytate, is an important plant
constituent. Phytic acid has six 'phosphate covalent
bond groups' that account for up to 85% of total
phosphorous in many cereals and legumes. Phytic
acid has 12 replaceable protons in the phytic
molecule, giving it the ability to complex with multi-
valent cations and positively charged proteins. Tsao
et al. (1997) studied the adsorption of heavy metal
ions by immobilized phytic acid. It was found that
244 Short communication
the immobilized phytic acid can adsorb heavy metal
ions, such as cadmium, copper, lead, nickle and zinc,
from aqueous solutions.
Although many studies have indicated the wide
existence of phytate-metal complexes in nature,
phytic acid has not been considered as an adsorbent
for the removal of heavy metal ions from crude
fermentative substrates.
The object of this study was to investigate the
potential of employing phytate as conversion-
enhancing agent for citric acid production from
untreated beet molasses by Aspergillus niger.
METHODS
Microorganism
Aspergillus niger W1-2, was isolated in our labora-
tory. It was maintained on malt extract slants and
stored at 4°C and subcultured every other month
(Zhou & Wang, 1992).
Medium
Beet molasses, obtained from a local sugar mill, was
used as substrate. The metal composition of this
molasses is given in Table 1. For the preparation of
fermentation medium, the molasses was diluted with
distilled water to adjust the sugar concentration. The
medium for citric acid production contained the
following (g/l): molasses (total reducing sugar), 100;
NHaCI, 4.0; KH2PO4, 1"0; MgSO4.7HaO, 0.25. The
pH of the medium was adjusted to 4"0.
Analytical methods
The fermentation broth was filtered through filter
paper in order to remove mycelia. The filtrate was
analyzed. Citric acid was determined by the coiori-
metric method of Marrier & Boulet (1958).
Reducing sugar was analyzed by the DNS
(3,5-dinitrosalicyclic acid) method (Miller, 1959).
Mycelial concentration was estimated by means of
dry weight measurement. Reducing sugars were
determined as glucose.
Fermentation conditions
Fermentations were carried out in 250-ml Erlen-
meyer flasks containing 50 ml fermentation medium,
incubated at 30°C and 200 rev/min in an orbital
shaker (Wang & Liu, 1998). Sodium phytate was
supplemented to the fermentation medium to
investigate the effect of phytate on citric acid
production. All experiments were conducted in three
Table 1. Metal ions of molasses used in this study
Concentration (g/kg) Na K Ca
8.5 26-4 5-8
replicates. The results given are the mean value of
all tests.
RESULTS A N D D I S C U S S I O N
Experiments were carried out adding various
concentrations of phytate into the medium at two
different stages of fermentation in otherwise
identical fermentations in order to investigate the
influence of phytate on citric acid production. The
phytate concentration varied from 4 g/l to 20 g/1 in
the fermentation medium. The results are shown in
Fig. 1.
Figure 1 shows the citric acid production with
different concentrations of phytate added at two
different stages of fermentation. The addition of
phytate, as an ion-exchanger resulted in an improve-
ment in citric acid production. The effect of phytate
addition was dependent on concentration and the
stage of fermentation at which phytate was added.
When added at the beginning of incubation, the
optimal concentration of phytate in the medium for
citric acid production was 10 g/l, which resulted in
about a 3"l-fold increase in citric acid accumulation.
During the fermentation, addition of 16 g/1 phytate
to the medium during the fermentation after 3 days
incubation gave the maximal citric acid production,
which was about 2.4-fold higher than in the control
experiment (no phytate). The effect of phytate was
more pronounced when added at the fermentation
stage than added at the beginning of cultivation.
The comparison of citric acid fermentation with
or without addition of 10 g/1 phytate at the beginning
of cultivation is shown in Fig. 2.
From Fig. 2, it could be concluded that the
addition of phytate caused increase of reducing
sugar utilization and enhanced citric acid accumula-
tion, however, the mycelial growth was slightly
inhibited.
The stimulation of citric acid production by
phytate addition can be explained in terms of a
favourable adjustment of trace metal balance in the
broth. The extent of the effect produced will depend
on the metal ion concentrations of the molasses, the
effect of the complexing agent on the fermentative
microorganism and the influence of other constit-
uents of the molasses.
Phytate is a natural material that can be found in
the seeds of cereals and legumes. One of the most
readily available sources of phytate is corn steep
liquor, a by-product from corn wet milling. In a
typical concentrated corn steep liquor from corn wet
milling process there is about 4% of phytate on a
Metal ions
Mg Zn Fe Mn Cu
0.072 0.11 0.08 0.022 0'001
 Short communication 245

support of this work (Research grant agreement No.

E/2546-1).
6O

REFERENCES

1 40 Choudhary, A. O. & Pirt, S. J. (1966). The influence of

metal-complexing agents in citric acid production by
Aspergillus niger. J. Gen. Microbiol., 43, 71-78.
Clark, D. S., Ito, K. & Tymchuk, P. (1965). Effect of
20 potassium ferrocyanide on the chemical composition of
molasses used in citric acid fermentation. Biotechnol.
Bioengng, 7, 269-275.
Ergun, M., Mutlu, S. F. & Gurel, O. (1997). Improved
0 I ,, I I I
ethanol production by Saccharomyces cerevisiae with
0 4 8 12 16 20 EDTA, ferrocyanide and zeolite X addition to sugar
Phytate concentration (g/L) beet molasse~ J. Chem. Tech. Biotechnol., 68, 147-150.
Marrier, J. R. & Boulet, M. (1958). Direct determination
Fig. 1. Effect of phytate on citric acid production from of citric acid in milk with improved pyridine-acetic
beet molasses by Aspergillus niger when added at growth anhydride method. J. Dairy Sci., 41, 1683-1692.
stage (0 h) (~) and fermentative stage (72 h) ([]). Miller, G. L. (1959). Use of dinitrosalycilic acid reagent
for determination of reducing sugars. Anal Chem., 31,
dry basis. Therefore, phytate can be a low-cost and 426-429.
readily available material suitable for stimulation of Oderinde, R. A., Ngoka, L. C. & Adesogan, E. K. (1986).
citric acid fermentations. Comparative study of the effect of ferrocyanide and
EDTA on the production of ethyl alcohol from molasses
by Saccharomycs cerevisiae. Biotechnol. Bioengng, 28,
ACKNOWLEDGEMENTS 1462-1465.
Oderinde, R. A., Okogun, J. I. & Esuoso, K. O. (1990).
The influence of CDTA and DPTA on ethanol produc-
The author gratefully acknowledges the Inter- tion from sugar cane molasses using Saccharomyces
national Foundation for Science (IFS) for financial cerevisiae. Die Nahrung, 34, 171-175.
Tsao, G. T., Zheng, Y., Lu, J. & Gong, C. S. (1997).
Adsorption of heavy metal ions by immobilized phytic
acid Appl. Biochem. Biotechnol., 63, 731-741.
Wang, Jianlong & Liu, Ping (1996). Comparison of citric
•~ 6O
acid production by Aspergillus niger immobilized in gels
2 and cryogels of polyacrylamide. J. Ind. Microbiol., 16,
351-353.
Wang Jianlong & Liu Ping (1998). Phytate as a stimulator
"~ 40 to enhance the citric acid production by Aspergillus
niger. Process Biochem., 33, 313-316.
Zhou, Ding & Wang, Jianlong (1992). Study on breeding
of citric acid-producer and its fermentation• Food
Ferment. Industries, 5, 45-49 (in Chinese).

~ 0 --
j/ 2
1 2
Fig. 2. Comparison of citric acid production with or
without adding 10 g/1 phytate at the beginning of cultiva-
tion. (1)Reducing sugar; (2) citric acid; (3) mycelial
weight (m) with added phytate; ([]) without added phytate.
Jianlong Wang
State Key Joint Laboratory of Environment Simulation and
Pollution Control, Department of Environmental Science &
3 Engineering, Tsinghua University, Beijing 100084,
People's Republic of China
(Received 12 December 1997; revised version received
16 January 1998; accepted 21 January 1998)