Bioresource Technology 93 (2004) 125–130

Citric acid production by selected mutants of Aspergillus niger

from cane molasses
a,* a,1
Haq Ikram-ul , Sikander Ali , M.A. Qadeer b, Javed Iqbal c

a
Biotechnology Research Laboratory, Department of Botany, G.C. University Lahore, Lahore 54000, Pakistan
b
Centre of Excellence in Molecular Biology, University of the Punjab, Thokar Niaz Baig, Lahore 54000, Pakistan
c
School of Biological Sciences, University of Punjab, Quaid-e-Azam Campus, Lahore 54000, Pakistan
Received 15 August 2002; received in revised form 28 October 2003; accepted 29 October 2003

Abstract
The present investigation deals with citric acid production by some selected mutant strains of Aspergillus niger from cane
molasses in 250 ml Erlenmeyer flasks. For this purpose, a conidial suspension of A. niger GCB-75, which produced 31.1 g/l citric
acid from 15% (w/v) molasses sugar, was subjected to UV-induced mutagenesis. Among the 3 variants, GCM-45 was found to be a
better producer of citric acid (50.0 ± 2a) and it was further improved by chemical mutagenesis using N-methyl, N-nitro-N-nitroso-
guanidine (MNNG). Out of 3,2-deoxy-D -glucose resistant variants, GCMC-7 was selected as the best mutant, which produced
96.1 ± 1.5 g/l citric acid 168 h after fermentation of potassium ferrocyanide and H2 SO4 pre-treated blackstrap molasses in Vogel’s
medium. On the basis of kinetic parameters such as volumetric substrate uptake rate (Qs ), and specific substrate uptake rate (qs ), the
volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing
organisms and produced more citric acid. The mutant GCMC-7 has greater commercial potential than the parental strain with
regard to citrate synthase activity. The addition of 2.0 · 10
5 M MgSO4 Æ 5H2 O into the fermentation medium reduced the Fe2þ ion
concentration by counter-acting its deleterious effect on mycelial growth. The magnesium ions also induced a loose-pelleted form of
growth (0.6 mm, diameter), reduced the biomass concentration (12.5 g/l) and increased the volumetric productivity of citric acid
monohydrate (113.6 ± 5 g/l).

2003 Published by Elsevier Ltd.

Keywords: Citric acid; Blackstrap molasses; Mutation; Aspergillus niger; Mg2þ ions; Fe2þ ions reduction; Mould morphology

1. Introduction (Gupta and Sharma, 1995). Owing to the steadily

Citric acid fermentation is one of the largest bio-
technological industries. Citric acid is used in several
industries (Rohr, 1998). According to the estimates, ci-
tric acid produced through fermentation is 7.0 · 105 ton/
annum (Ali et al., 2001). Aspergillus niger has the po-
tential to produce a number of primary and secondary
metabolites. The techniques of ultraviolet irradiations,
gamma rays or N-methyl, N-nitro-N-nitroso-guanidine
(MNNG) induced mutagenesis are useful to improve
the yield of various secondary metabolites by A. niger
*
Corresponding author.
E-mail addresses: ikrhaq@yahoo.com (H. Ikram-ul), alisbiotech@
yahoo.com (S. Ali).
1
Present address: Department of Plant Soil Science and General
Agriculture, Southern Illinois University at Carbondale, Carbondale,
IL 62901, USA.
increasing demand of citric acid for industrial purposes,
its manufacture from cane or beet molasses has proved
to be of great importance to the sugar industry (Pazouki
et al., 2000).
Citric acid produced by A. niger is extremely sensitive
to trace metals present in molasses such as iron, zinc,
copper and manganese, etc. especially during submerged
fermentation. The concentration of these heavy metals,
should be decreased well below that required for optimal
mycelial growth (Majolli and Aguirre, 1999). The opti-
mum concentration of Feþþ required for citric acid
production has been found to vary with the strain of the
fungus and cultural conditions employed. It has been
reported (Pera and Callieri, 1997) that citric acid pro-
duction by A. niger using molasses as the substrate is
severely affected by the presence of iron at a concen-
tration as low as 0.2 ppm (w/v). However, addition of
magnesium at about 0.1–20 ppm (w/v) at the time of

0960-8524/$ - see front matter
2003 Published by Elsevier Ltd.

doi:10.1016/j.biortech.2003.10.018
126 H. Ikram-ul et al. / Bioresource Technology 93 (2004) 125–130
inoculation or during the first 48 h of fermentation was
found to counter-act the deleterious effect of iron. It was
found that the addition of MgSO4 Æ 5H2 O at 1.02 mg/l
cane molasses resulted in a better conversion of sugar
into citric acid (Fedoseev, 1970).
In the present study, we report the isolation of
mutants of A. niger for hyper production of citric acid
through UV-irradiation followed by MNNG-muta-
genesis. The best mutants were compared with parental
strain for citric acid production following fermentation
of blackstrap molasses. The study revealed that addition
of magnesium ions have a direct influence on mycelial
morphology, provoked pellet formation, reduced Fe2þ
concentration in molasses and consequently increased
volumetric citric acid productivity.
2. Methods
2.1. Organism and culture maintenance
A. niger strain GCB-75 was collected from the culture
collection of Biotechnology Research Laboratories,
Department of Botany, Government College University
Lahore, Pakistan. It was maintained on potato dextrose
agar slants and stored at 4 C in a refrigerator. All the
culture media, unless otherwise stated, were sterilized at
15-lb/in.2 pressure (121 C) for 15 min.
2.2. Inoculum development
2.2.1. Preparation of conidial inoculum
The conidial inoculum was used for ultraviolet irra-
diation as well as optimisation by shake flask technique.
Conidia from 4 to 6 day old slant culture were used for
inoculation. Ten ml of sterilized 0.005% (w/v) Monoxal
O.T. (Diacetyl ester of sodium sulfo succinic acid) was
added to the slant having profuse conidial growth on its
surface. Inoculating needle was used for breaking the
clumps of conidia and the test tube was shaken vigor-
ously to obtain a homogenous mixture of conidial sus-
pension. The conidial count was 6.2 · 106 conidia/ml.
2.2.2. Preparation of vegetative inoculum
Vegetative inoculum was used for chemical mutation
as well as scale-up studies in stirred fermentor. A volume
of 45.0 ml of Vogel’s medium (containing w/v, 0.5%
trisodium citrate, 0.5% KH2 PO4 , 0.2% NH4 NO3 , 0.4%
(NH4 )2 SO4 , 0.02% MgSO4 , 0.1% peptone, 0.2% yeast
extract and pH 5.5) was dispensed in 250 ml conical
flask. Chromic acid washed marble chips (12–15 in
number) were added in the flask (to break up the
mycelial pellets) and were sterilized. Two ml of 50% (w/
v) stock solution of glucose were aseptically added into
the autoclaved Vogel’s medium as a carbon source. The
flasks were inoculated with 1.0 ml A. niger conidia under
aseptic conditions. Inoculum was allowed to grow on 30
C in a rotary incubator shaker (160 rpm). The cells
were harvested, centrifuged at 9000 rpm (8331g for 15
min), washed twice with saline water and re-suspended
in saline solution. The optical absorbance was measured
spectrophotometrically (at 610 nm wavelength) and
maintained at 0.5 · 10 dilution. This mycelial suspension
was used for mutational work or to inoculate the citric
acid fermentation medium.
2.3. Induction of mutation
For ultraviolet irradiation, the conidial suspension of
24 h old slant culture of A. niger GCB-75 in saline water
was transferred to sterile 0.005% (w/v) dioctyl sulfo
succinate (Sigma Chemical Company, St. Louis, USA)
solution. The colonies forming units/ml (CFU/ml) on
oxgall-potato-dextrose-agar medium were maintained at
6.2 · 106 cells/ml for UV-irradiation and further studies.
The dose of UV exposure to suspension was 1.2 · 106 J/
m2 /S for different time intervals (15–180 min). Survival
curve was prepared and time of exposure giving
(0.5 · 106 CFU/ml) 3log-kill was selected for mutation of
the organism. The mutant derivatives were selected (120
mutants), characterized in liquid culture media as de-
scribed previously (Parvez et al., 1998). The best mutant
was selected from a number of variants and designated
as GCM-45. The chemical mutagenesis was carried out
by using MNNG, following the method of Roy and Das
(1978). Ninety-one mutants were isolated. The best
mutant, which exhibited enhanced production of citric
acid in plate and liquid culture, was designated as
GCMC-7.
2.4. Fermentation technique
2.4.1. Shake flask technique
All the shake flask studies of citric acid fermentation
were carried out using the medium composed of potas-
sium ferrocyanide + H2 SO4 clarified blackstrap molasses
in Vogel’s medium (Parvez et al., 1998). Initial pH of
fermentation medium was adjusted at 5.5. For sub-
merged fermentation, 190 ml of the medium was dis-
pensed in 1000 ml conical flasks and sterilized. After
cooling at room temperature, the flasks were inoculated
with 10% (v/v) inoculum containing 6.2 · 106 CFU/ml in
triplicate and incubated on an orbital shaker (200 rpm)
at 30 C for different time intervals (12, 24, 48, 72, 96,
120, 144, 168 and 192 h). All the shake flask experiments
were conducted in triplicates.
2.4.2. Fermentor study
Vegetative inoculum was developed according to the
method of Khan et al. (1970) as described earlier. An
agitator bioreactor (GLSC-AF-199-10) of 9-l working
capacity (total capacity 15-l) was employed for all
 H. Ikram-ul et al. / Bioresource Technology 93 (2004) 125–130
microbial cultivations. Fermentation medium consist-
ing of clarified cane molasses (Kamalia Sugar Mills,
Kamalia, Pakistan) containing (% w/v): Sugar 15.0,
NH4 NO3 0.025, ash contents 0.45, trace metals like iron,
zinc, aluminium 0.035 and K4 Fe(CN)6 200 ppm, w/v
(initial pH 6.0) was sterilized at 15 lb/in.2 pressure (121
C) for 20 min. The inoculum was used at a level of
4.0% (v/v). Incubation temperature was kept at 30 C
throughout the fermentation period of 144 h. Aeration
rate was kept at 1.0 l/l/min (35% dissolved O2 level).
Sterilized silicone oil, 10%, v/v (antifoam AE-II) was
used to control foaming during the fermentation. Sam-
ples were taken after every 24 h.
2.5. Assay methods
2.5.1. Sugar
Sugar was estimated gravimetrically by DNS method
(Ghose, 1969). A double beam UV/Vis scanning spec-
trophotometer (Model: Cecil-CE 7200-series, Aquarius,
UK) was used for measuring the % colour intensity.
2.5.2. Dry cell mass
Dry cell mass was determined by filtering the culture
medium through weighed Whattman filter paper no. 44.
The filtrate was used for further analysis. For the cal-
culation of dry cell mass, mycelium was thoroughly
washed with tap water and dried at 105 C for 2 h in an
oven (Model: 1442A, Memmort, Germany) following
the method of Haq and Daud (1995).
2.5.3. Total acid
Total acid was estimated by titrating 10.0 ml of
diluted culture filtrate against 0.1 N NaOH using
phenolphthalein as an indicator. The total acid was
reported in g/l.
% Total acid
Titre  normality of alkali  equivalent weight of acid
¼
Volume of sample  1000
 100
The equivalent weight of the acid is 70.
2.5.4. Citric acid
Citric acid was estimated gravimetrically, using pyri-
dine–acetic anhydride method as reported by Marrier
and Boulet (1958). One ml of the diluted culture filtrate
along with 1.30 ml of pyridine was added in the test tube
and swirled briskly. Then 5.70 ml of acetic anhydride
was added in the test tube. The test tube was placed in a
water bath at 32 ± 0.25 C for 30 min. The optical
density was measured on a spectrophotometer (405 nm)
and citric acid contents of the sample were estimated
with reference (run parallel, replacing 1.0 ml of the
culture filtrate with distilled water) to the standard. The
127
% of citric acid was determined on the basis of sugar
used.
2.5.5. Ferrocyanide ions
Ferrocyanide was estimated by the method of Mar-
rier and Clark (1962). Five ml of the culture filtrate was
transferred to the test tube. Two ml of 50% (w/v) citric
acid solution and 1.0 ml of ferric chloride reagent were
added to it. The contents were mixed thoroughly, set
aside for 60–90 min at room temperature. A blank
was also run parallel with 5.0 ml of distilled water in
place of ferrocyanide dilution, followed by the above
procedure. The optical density was measured at 690 nm
using spectrophotometer. To correct for dark colour
of molasses, transferred a duplicate aliquot of sam-
ple solution to another colorimeter tube and continued
as described above but substituted ferric chloride re-
agent with 1.0 ml of distilled water. Subtracted the
optical density of this sample measured against a nor-
mally prepared reagent blank from that of the test
solution.
2.6. Kinetic parametric studies and statistical analysis
Kinetic parameters for batch fermentation process
were determined after Pirt (1975). Treatment effects were
compared after Snedecor and Cochran (1980). Signifi-
cance has been presented as Duncan’s multiple ranges in
the form of probability (hpi) values.
3. Results and discussion
In the present study, a wild culture of A. niger (GCB-
75) and its mutant derivatives were examined for the
production of citric acid from 15.0% (w/v) carbohy-
drates using cane molasses in 1000 ml Erlenmeyer flasks
(Table 1). The parental strain and best mutants (GCM-
45 and GCMC-7) consumed 36.0 ± 2.5, 110.0 ± 1.5,
120.0 ± 2.3 g/l sugars and synthesized 31.1 ± 2.0, 50.0 ±
2.0, 96.1 ± 1.5 g/l citric acid, respectively. These strains
also synthesized 16.0 ± 1.4, 28.9 ± 0.4, 28.9 ± 0.4 g/l dry
cell mass. All the mutants were significantly improved
for the value of Yx=s over the parental strain. Maximum
growth in terms of specific growth rate was only mar-
ginally different during growth of the wild parent and its
two mutant derivatives. However, when the cultures
were monitored for Yp=s and Yp=x , there was significant
enhancement (p 6 0:05) in these variables in mutant
cultures over those obtained for wild culture of A. niger.
The kinetic parameters are presented in Table 2. A. niger
mutants exhibited improved production of citric acid
over the parental strain. Maximum values for Qp , Qs and
qp were several-fold improved over those from some
other A. niger cultures or mutants (Parvez et al., 1998;
Snedecor and Cochran, 1980; Bennet and Klich, 1992;
128 H. Ikram-ul et al. / Bioresource Technology 93 (2004) 125–130

Table 1
Citric acid production by UV-induced and N-methyl, N-nitro-N-nitroso-guanidine treated mutant strains of A. niger following growth on potassium
ferrocyanide pre-treated cane molasses (15% w/v, carbohydrates) using Vogel’s medium (pH 5.5) in 1000 ml Erlenmeyer flasks
Mutant strains tested Citric acid (g/l) Product and growth yield coefficients
Yx=s (g/g) Yp=s (g/g) Yp=x (g/g)
Parental strain
GCB-75 31.1 ± 2d 0.39 ± 0.03c 0.45 ± 0.04a 1.9 ± 0.1d
UV-induced
GCM-35 42.0 ± 3c 0.42 ± 0.02b 0.40 ± 0.03b 2.1 ± 0.20c
GCM-45 50.0 ± 2a 0.44 ± 0.04ab 0.44 ± 0.03a 2.6 ± 0.20a
GCM-55 45.0 ± 3b 0.45 ± 0.03a 0.45 ± 0.03a 2.4 ± 0.2b
MNNG-induced
GCMC-45 50.0 ± 2d 0.44 ± 0.04b 0.65 ± 0.04d 2.6 ± 0.20d
GCMC-2 65.0 ± 4c 0.45 ± 0.03b 0.75 ± 0.04c 2.8 ± 0.20b
GCMC-4 75.0 ± 5b 0.46 ± 0.03b 0.80 ± 0.05b 2.7 ± 0.20c
GCMC-7 96.1 ± 1.5a 0.49 ± 0.04a 1.0 ± 0.01a 3.3 ± 0.2a
Cultural conditions: temperature 30 C, initial sugar concentration 150 g/l, initial pH 6.0. Each value is an average of three replicates. Yx=s ¼ g cells/g
substrate utilized, Yp=s ¼ g citric acid produced/g substrate consumed, Yp=x ¼ g citric acid produced/g cells. ± Denotes standard deviation among the
replicates. Numbers followed by letters differ significantly at p 6 0:05 within each column.

Table 2
Kinetic parameters for the production of citric acid from sugars in molasses following growth of A. niger and its mutant derivatives
Kinetic parameters Parental strain GCB-75 Mutant GCM-45 Mutant GCMC-7
Specific growth rate
l (h
1 ) 0.20 ± 0.02a 0.23 ± 0.02a 0.26 ± 0.02a
Substrate consumption parameters
Qs (g/l/h) 0.38 ± 0.02c 0.47 ± 0.03b 0.93 ± 0.04a
qs (g/g cells/l/h) 0.51 ± 0.03a 0.52 ± 0.03a 0.53 ± 0.04a
Qx (g cells/l/h) 0.33 ± 0.03c 0.39 ± 0.03b 0.47 ± 0.04a
Citric acid formation parameters
Qp (g/l/h) 0.38 ± 0.03b 0.39 ± 0.03b 0.74 ± 0.04a
qp (g/g cells/h) 0.38 ± 0.02c 0.60 ± 0.04b 0.83 ± 0.05a
Cultural conditions: temperature 30 C, initial sugar concentration 150 g/l, initial pH 6.0. Each value is an average of three replicates. ± Shows
standard deviation among the replicates. Qs ¼ g substrate consumed/l/h, qs ¼ g substrate consumed/g cells/h, Qx ¼ g cells formed/l/h, Qp ¼ g citric
acid produced/l/h, qp ¼ g citric acid produced/g cells/h. The values within each column differ significantly at p 6 0:05.

Kirimura et al., 1992). Succinic acid, malic acid and
fumaric acid were also produced but their highest vol-
umetric productivities, in mutant GCMC-7, were 0.14,
0.13 and 0.1 g/l/h only.
The effect of different magnesium sources [MgSO4 Æ
5H2 O, MgCl2 , Mg(NO3 )2 and MgCO3 ] and their con-
centration on the production of citric acid by the
parental strain of A. niger GCB-75 and its mutant
derivative GCMC-7 in agitator bioreactor was carried
out (Table 3). It was observed that 2.0 · 10
5 M
MgSO4 Æ 5H2 O gave maximum production of citric acid
monohydrate (112.05 g/l). This might be due to the fact
that MgSO4 Æ 5H2 O gave free Mg2þ ions in the produc-
tion medium which were not only beneficial for mycelial
growth but also counter-act the deleterious effects of
Fe2þ on fungal development. The presence of Mg2þ ions
in the medium also induced pellet like morphology of
mycelium. The enhancement in citric acid production
was substantial (Fig. 1). At low concentration of Mg2þ
ions (1.0 · 10
5 M), production of citric acid was also
low (84.6 ± 3 g/l). The presence of MgSO4 Æ 5H2 O con-
centration higher than 2.0 · 10
5 M did not have any
major effect on the structure and external aspects of the
pellets but affected citric acid production which de-
creased significantly (81.5 ± 3 g/l, 70.6 ± 2 g/l). The
results presented show that pellets with a suitable
structure and morphology improved citric acid pro-
duction. Some authors believed that Mg2þ may stimu-
late citric acid production by inhibiting aconitase
(Sanjay and Sharma, 1994) others reject this possibility,
finding no alterations in the citrate: isocitrate ratio on
adding Mg2þ under the conditions of citric acid pro-
duction. However, they observed favourable changes in
citric acid yield (Shoukat et al., 1997).
The cultural conditions influence the growth pattern
of filamentous fungi, which can range from a pellet to a
dispersed filamentous form, affecting in this way both
the growth rate and product formation. The apparent
viscosity of a culture growing in the pellet form is lower
than that corresponding to a filamentous form. There-
 H. Ikram-ul et al. / Bioresource Technology 93 (2004) 125–130 129

Table 3
The influence of different magnesium sources and their concentration on the production of citric acid by A. niger GCB-75 (parental strain) and
GCMC-7 (mutant strain) in a agitator bioreactor
Magnesium Concentration Citric acid Sugar Dry cell mass (g/l) Mycelial morphology
sources of magnesium monohydrate (g/l) consumption (g/l)
sources GCB-75 GCMC-7 GCB-75 GCMC-7 GCB-75 GCMC-7 GCB-75 GCMC-7
(a  10
5 M)
Control – 32.4 ± 3 75.4 ± 4 95.20 86.50 9.00 10.20 Small pellets Intermediate
pellets
Magnesium 1.0 · 10
5 33.8 ± 1 84.6 ± 3 91.50 88.20 9.50 13.00 Mixed pellets Small pellets
sulphate 2.0 · 10
5 39.4 ± 2 112.0 ± 5 89.20 84.60 10.50 12.50 Mixed pellets Mixed pellets
3.0 · 10
5 38.2 ± 2 81.5 ± 3 87.50 82.02 12.40 16.20 Laxy pellets Mixed pellets
4.0 · 10
5 31.0 ± 5 70.6 ± 2 86.20 81.20 9.20 12.00 Small pellets Small pellets
Magnesium 1.0 · 10
5 26.1 ± 3 51.6 ± 2 91.50 90.20 7.00 8.60 Large pellets Last mycelia
chloride 2.0 · 10
5 29.0 ± 4 59.0 ± 1 105.50 91.60 9.80 10.60 Broken pellets Small pellets
3.0 · 10
5 21.1 ± 3 54.2 ± 2 108.20 87.60 9.50 12.00 Gummy mass Small pellets
4.0 · 10
5 18.2 ± 3 52.5 ± 3 101.40 86.00 7.60 14.00 Gummy mass Small pellets
Magnesium 1.0 · 10
5 25.5 ± 4 61.0 ± 4 112.50 101.60 12.10 11.80 Viscous Gummy mass
nitrate 2.0 · 10
5 27.4 ± 4 65.6 ± 4 116.80 109.80 11.60 10.80 Viscous Gummy mass
3.0 · 10
5 18.8 ± 1 64.2 ± 4 121.60 110.80 9.20 9.60 Viscous Gelatinous
4.0 · 10
5 12.4 ± 2 62.8 ± 3 102.80 106.50 9.90 9.05 Viscous Gelatinous
Magnesium 1.0 · 10
5 29.8 ± 2 41.2 ± 5 100.20 100.05 15.05 13.20 Gummy mass Viscous
carbonate 2.0 · 10
5 34.2 ± 3 56.0 ± 4 96.20 101.80 9.40 10.60 Fluffy mycelia Viscous
3.0 · 10
5 31.2 ± 2 50.4 ± 2 81.60 92.60 9.00 9.80 Fluffy mycelia Viscous
4.0 · 10
5 29.0 ± 1 45.2 ± 3 80.20 83.60 8.06 9.30 Gelatinous Fine pellets
Cultural conditions: temperature 30 C, aeration rate 1.0 l/l/min, initial sugar concentration 150 g/l, initial pH 6.0. Each value is an average of three
parallel replicates. The symbol ± shows the standard deviation among the replicates. The values differ significantly at p 6 0:05 within each column.

135 6
5.8
6 branched hyphae favours the formation of pellets, thus
improving the performance of the process (Pera and
Dry cell mass, Sugar consumed, Citric

5.5
120
5.1
5
Callieri, 1997; Haq et al., 2001). It was observed that in
105
the medium without Mg2þ (Blank, Table 3), the mycelial
90 4.3 formation stages were not fulfilled. The pellets formed
4
were less in number (20 pellets/ml) and irregular in form
pH change
acid (g/l)

75
60
45
30
15
0
0 24 48
Dry cell mass (g/l)
Fig. 1. Rate of citric acid fermentation by the mutant strain A. niger
GCMC-7 using cane molasses as the basal substrate. Cultural condi-
tions: temperature 30 C, aeration rate 1.0 l/l/min. Magnesium sul-
phate (2.0 · 10
5 M) was added 6 h after the fermentation. Each value
is an average of three parallel replicates. Y -error bars show the stan-
dard deviation among the replicates. The values in each set differ sig-
nificantly at p 6 0:05.
fore, the quantitative relation between both forms of
growth influences the over-all rheological properties of
the culture (Kirimura et al., 1992). The positive effect of
Mg2þ might also be related to the increase of mycelial
branching level. The presence of shorter and highly
3.6
(0.3–1.0 mm in diameter). As desired, fluffy loose and
3 round pellets (0.6 mm, diameter) were formed by adding
2.8
2.0 · 10
5 M, MgSO4 Æ 5H2 O. Citric acid concentration
2.4
2.3
2
reached maximum (113.6 ± 5 g/l), 6 h after the incuba-
tion, which was very significant for experiments in agi-
tator bioreactor (Table 4). The dry cell mass (16.5 g/l)
1
72 96 120 144 168 192
was slightly higher in this case, as compared with the
Incubation period (h)
parental culture of A. niger. Growth was in the form of
Sugar consumed (g/l) Citric acid (g/l) pH
mixed laxy pellets.
From the work, it can be concluded that the presence
of Mg2þ ions in the fermentation medium is very
important in order to enhance a suitable pellet structure
(fluffy centre and lax surface) related to major cellular
physiology in citric acid fermentation by mutant strain
of A. niger GCMC-7. Magnesium ions reduce the con-
centration of free Fe2þ by making complexes such as
FeS and FeSO4 , which were precipitated out during the
course of fermentation. The mutational work in this
study has given a potent organism for production of
citric acid from blackstrap molasses (GCMC-7). More
work is, however, needed to improve the substrate
consumption rate by isolating mutants, which are
resistant to higher 2-deoxy-D -glucose levels.
130 H. Ikram-ul et al. / Bioresource Technology 93 (2004) 125–130

Table 4
Effect of time of addition of magnesium sulphate on citric acid production and mycelial morphology by parental strain of A. niger and its mutant
derivative in agitator bioreactor
Time of addition of Citric acid monohydrate (g/l) Mycelial morphology
MgSO4 Æ 5H2 O (h) GCB-75 GCMC-7 GCB-75 GCMC-7
Control 35.5 ± 2 78.2 ± 4 Mixed pellets Intermediate round pellets
0 38.6 ± 2 84.0 ± 3 Mixed pellets Intermediate round pellets
6 42.8 ± 3 113.6 ± 5 Mixed pellets Mixed laxy pellets
12 31.5 ± 4 88.4 ± 3 Large pellets Mixed pellets
18 30.4 ± 2 81.0 ± 2 Large pellets and elongated hyphae Large pellets
24 30.2 ± 3 76.8 ± 4 Gummy mass Large broken pellets
Cultural conditions: magnesium sulphate concentration 2.0 · 10
5 M, sugar added 150 g/l, agitation intensity 200 rev/min. Values differ significantly
at p 6 0:05 within each column. The sign ± indicates standard deviation among three parallel replicates.

Acknowledgements
This work is a part of the research project. Authors
acknowledge Pakistan Science Foundation, Islamabad
for financing the research project no. PSF/GC/Bio/Res
(283).
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