Int. J. Agric. Appl. Sci. Vol. 5, No.

1, 2013
8

CITRIC ACID PRODUCTION FROM SUGARCANE MOLASSES BY ASPERGILLUS NIGER UNDER DIFFERENT
FERMENTATION CONDITIONS AND SUBSTRATE LEVELS

Umar Farooq, Faqir Muhammad Anjum*, Tahir Zahoor*, Sajjad-ur-rahman**, Zafar Hayat***, Kashif Akram and
Ejaz Ashraf***
Institute of Food Science and Nutrition, University of Sargodha, Sargodha, Pakistan
*National Institute of Food Science and Technology, University of Agriculture, Faisalabad, Pakistan
**Department of Veterinary Microbiology, University of Agriculture, Faisalabad, Pakistan
***University College of Agriculture, University of Sargodha, Sargodha, Pakistan
Corresponding authors e-mail: umarfarooq@uos.edu.pk

ABSTRACT

Aspergillus niger, one of the most important fungi used in industrial microbiology, has been employed for many
years for the commercial production of citric acid. However, citric production and recovery may vary greatly
depending upon type of substrate as well as the fermentation conditions like temperature, fermentation time and
the type of culture/strain. The present study was aimed to investigate the potential of Aspergillus niger to convert
sugarcane molasses into citric acid through fermentation carried out for 8 days at three different temperatures
20C, 24C and 28C and five substrate levels 0, 6%, 12%, 18% and 24%. The optimum citric acid production was
achieved after 6 days of fermentation from 24% substrate level at 28C. The maximum citric acid yield was
6.870.12 g 100 mL
-1
(68.7 g L
-1
) with a mean recovery of 51.62% with respect to initial total sugar contents of the
media. However, the optimum recovery of citric acid (59.64%) with respect to initial total sugar contents was
achieved from 18% substrate level after the completion of fermentation period of 6 days. The study suggested that
the 6 days of fermentation period with 18% substrate level and temperature 28

C were the optimum conditions for

citric acid recovery from sugarcane molasses through Aspergillus niger.

Key words: Fermentation time, industrial microbiology, organic acids, substrate level, substrate type, waste
management

INTRODUCTION

The organic acids are extensively used in a variety of
food products as preservatives, pH adjuster,
sweetness enhancer, leavening agents and
stabilizers (Majumder et al., 2010). The acceptability
of food products mainly depends on the flavor
components, which are complex

as well as type-
specific. These flavor components are influenced

by
the presence of organic acids and other substances
like sulphur compounds,

lactones, methyl ketones,
alcohols and phenolic substances

(Urbach, 1993).
The important flavor

substances are formed as a
result of the hydrolysis

of fatty acids or by the
bacterial

growth, or enhanced by the addition of
acidulants during processing

(Adda et al., 1982). The
principal organic acids having food applications are
phosphoric, fumaric, tartaric, lactic and citric acids
(Berry, 2001). The citric acid is an important
commercial product, its global production has
reached to 1.7 million tons per year and its annual
increasing growth rate is 5% (Kana et al., 2012). The
largest amount of citric acid is consumed in food
industry using almost 70% of the total production,
followed by about 12% in the pharmaceutical
industry and 18% for other applications (Rodrigues
et al., 2013)

The production of citric acid by Aspergillus niger is
one of the most commercially utilized examples of
fungal overflow metabolism. Many microorganisms
such as fungi and bacteria can produce citric acid.
The various fungi, which have been found to
accumulate citric acid in their culture media, include
strains of Aspergillus niger, Aspergillus awamori,
Penicillium restrictum, Trichoderma viride, Mucor
piriformis and Yarrowia lipolytica (Arzumanov et al.,
2000). However, Aspergillus niger is considered as
the organism of choice for the production of citric
acid because of the fact that this organism has the
capacity to utilize varieties of substrates due to its
well developed enzymatic system (Munshi et al.,
2013). Currently, researchers and scientists are
engaged in exploring the suitability of various food
U. Farooq, F.M. Anjum, T. Zahoor, S. Rahman, Z. Hayat, K. Akram and E. Ashraf
9

waste materials for bioconversions which might be
the cheaper sources for the production of various
fermented and value added products. Among
various food industrial wastes, the sugarcane
molasses might be a desirable raw material for citric
acid fermentation due to its availability at relatively
low price and it can be the basic substrate for citric
acid fermentation using submerged technique of
fermentation (Pazouki et al., 2000).

The productivity of various fermented products is
greatly influenced by the type of substrate as well as
the fermentation conditions like temperature,
fermentation time and the type of culture/strain
(Ikram-ul-Haq et al., 2002). Keeping in view the
importance of citric acid and utilization of food
wastes to prevent from pollution, the present
research work was planned to optimize the
fermentation conditions for production of citric acid
by Aspergillus niger in stirred fermentor using
sugarcane molasses as the basal media.

MATERIALS AND METHODS

Collection of waste materials
The sugarcane molasses were collected from
Crescent Sugar Mills Ltd., Faisalabad, Pakistan.

Preparation of inoculum
The fungal spores from potato dextrose agar slants
were inoculated into the potato dextrose broth for
inoculum preparation and the broth was incubated
at 30C in orbital shaker by adding sterilized glass
beads into the inoculums flasks. The fungal biomass
produced after incubation for 48 hours was later on
used for fermentation.

Fermentation
The fermentation of sugarcane molasses was carried
out by Aspergillus niger at 20C, 24C and 28C with
0, 6%, 12%, 18% and 24% substrate levels. The
following media was used by the method of Jianlong
et al. (2000), with some modifications; while
composition of fermentation media for citric acid
was NH
4
Cl 0.4 g, KH
2
PO
4
0.1 g, MgSO
4
.7H
2
O 0.025 g
and sugarcane molasses (glucose was replaced with
molasses) 0, 6%, 12%, 18% and 24% (quantity of
ingredients for 100 mL
-1
)

Analysis of fermentation media
The fermentation medium was filtered through
Whattman filter paper and the filtrate was used for
the estimation of sugars and citric acid. The media
was analyzed on daily basis up to 8 days of
fermentation for the said parameters as described
below;

Estimation of sugars
Total sugars were estimated using Lane and Eynon
method through titration with Fehlings solution as
described by Kirk and Sawyer (1991).

Extraction of citric acid
The citric acid was recovered from fermented media
using calcium carbonate as recommended by Young
(1985).

Estimation of citric acid
The citric acid produced during fermentation was
determined by high performance liquid
chromatography; HPLC (Akalin et al., 2002) as
detailed below;

Sample preparation
During sample preparation, 7 mL of fermented
media was added to 40 mL of buffer-acetonitrile
mobile phase

(0.5% (w/v) (NH
4
)
2
HPO
4
(0.038 M) -
0.4% (v/v) acetonitrile

(0.049 M), at pH 2.24 with
H
3
PO
4
), extracted for 1 hour in orbital shaker and
centrifuged at 6000 x g for 5 min. The supernatant

was collected and filtered once through filter paper
Whattman No. 1 and twice

through a 0.45 m
membrane filter, and then used directly for HPLC
analysis.

Triplicate analyses were performed on all
samples.



HPLC Analysis
The analysis was carried out by HPLC with UV
detector (Perkin Elmer-series 200) at 214 nm using
RP-18 column (120 4.6 mm). The operating
conditions were:

mobile phase, aqueous 0.5% (w/v)
(NH
4
)
2
HPO
4
(0.038 M) - 0.2%

(v/v) acetonitrile (0.049
M) adjusted to pH 2.24 with H
3
PO
4
;

flow rate 0.3 mL
min
-1
; ambient column temperature. The mobile
phase was prepared by dissolving analytical-grade

(NH
4
)
2
HPO
4
in distilled deionized water, HPLC-grade
acetonitrile, and

H
3
PO
4
. HPLC-grade reagents were
used as standards (Sigma Chemical

Co., St. Louis,
MO). Solvents were filtered through a 0.45 m

membrane filter and degassed under vacuum.
Quantification was based

on internal standard
method.



Statistical analysis
The statistical analysis of the data were carried by
Int. J. Agric. Appl. Sci. Vol. 5, No.1, 2013
10

following three factor factorial experiment and
Duncans multiple range (DMR) tests were used to
separate treatment means at p0.05 by the software
Minitab according to the methods described in Steel
et al. (1997).

RESULTS

Utilization of total sugars
The utilization of total sugars was significantly
(p0.05) influenced by fermentation time, substrate
levels and fermentation time x substrate levels
during citric acid production by Aspergillus niger. The
effect of temperature on utilization of total sugars
was also found significant. The first order interaction
of all other variables showed non-significant
influence on utilization of total sugars except
interaction between fermentation time and
substrate levels (Table 1).

Table 1: Mean squares for utilization of total sugars and production of citric acid during fermentation

Source of variance
DF
Mean squares
Utilization of total sugars Citric acid production
Fermentation time (D) 8 287.42** 119.96**
Temperature (T) 2 0.22

** 0.10
NS

Substrate level (S) 3 311.86** 160.06**
D x T 16 0.012
NS
0.004
NS

D x S 24 23.03** 11.37**
T x S 6 0.01
NS
0.004
NS

D x T x S 48 0.002
NS
0.001
NS

Error 216 0.04 0.10
Total 323
**Highly significant (p0.01), *Significant (p0.05),
NS
Non-significant (p0.05)

The total sugars utilization was progressively higher
with increase in fermentation time during citric acid
fermentation of sugarcane molasses. The results
showed that the highest total sugars utilization was
occured at 8
th
day of fermentation with a mean
value of 7.02 g 100 mL
-1
while significantly lowest
utilization of total sugars was found at the start of
fermentation. However, the results for total sugars
utilization at 8
th
and 7
th
day of fermentation as well
as at 6
th
and 7
th
day of fermentation for utilization
showed non-significant difference (Fig. 1).

The utilization of total sugars was significantly
(p0.05) higher at 28C (4.67 g 100 mL
-1
) whereas,
significantly lower utilization of total sugars was
recorded at 20C (4.58 g 100 mL
-1
) (Fig. 2). By
comparing the effect of substrates, it was found that
there was an increasing trend in utilization of sugars
with an increment in the concentration of substrate
(Fig. 3). The interactive effect of fermentation time
and substrate levels indicated a linear trend in
utilization of total sugars after about 24 hr (1 day) of
fermentation and reached to a constant level after 2,
3, 4 and 5 days of fermentation in the media
containing 6%, 12%, 18% and 24% substrate levels,
respectively (Table 2). However, significantly
(p0.05) higher utilization of total sugars was
observed after 8 days followed by 7 days of
fermentation in the media containing 24% level of
substrate with mean values of 10.410.07 g 100 mL
-1

and 10.340.03 g 100 mL
-1
, respectively (Table 2).
Moreover, the results further showed that the per
day utilization of total sugars increased with an
increase in substrate level of 18% and 24% up to 3 to
4 days of fermentation and then it was leveled off
(Fig. 4).

Citric acid production
The analysis of variance given in Table 1 indicated
that the effect of fermentation time, substrate level
and that of first-order interaction between
fermentation time and substrate levels was highly
significant (p0.05) on citric acid production from
sugarcane molasses while interaction of all other
variables showed non-significant effect on citric acid
production. With an increment in fermentation time,
the citric acid production was also increased and
there was a linear relationship between
fermentation time and the productivity of citric acid
(Fig. 5). Significantly highest production of citric acid
was observed in the media containing 24% substrate
level with mean value of 4.35 g 100 mL
-1
followed by
the media containing 18% substrate with a mean
value of 3.84 g 100 mL
-1
(Fig. 6). However, the
U. Farooq, F.M. Anjum, T. Zahoor, S. Rahman, Z. Hayat, K. Akram and E. Ashraf
11

interaction of fermentation time and substrate levels
(Table 3) indicated that with increasing time of
fermentation and substrate level, the citric acid
production was increased. The citric acid production
after 2, 3, 4 and 5 days of fermentation in the media
containing 6%, 12%, 18% and 24% substrate levels,
respectively was stabilized. It was indicated the citric
acid production in the media containing substrate

0.00 H
0.85 G
2.41 F
4.56 E
6.18 D
6.74 C
6.89 B
6.98 AB
7.02 A
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
0 1 2 3 4 5 6 7 8
U
t
i
l
i
z
a
t
i
o
n

o
f

t
o
t
a
l

s
u
g
a
r
s

(
g

1
0
0

m
L
-
1
)
Fermentation time (days)

Fig. 1: Effect of fermentation time on utilization of total sugars during citric acid production from sugarcane
molasses


4.58 B
4.62 AB
4.67 A
4.52
4.54
4.56
4.58
4.60
4.62
4.64
4.66
4.68
20 24 28
U
t
i
l
i
z
a
t
i
o
n

o
f

t
o
t
a
l

s
u
g
a
r
s

(
g

1
0
0

m
L
-
1
)
Temperature (C)

Fig. 2: Effect of temperature on utilization of total sugars during citric acid production from sugarcane molasses

level of 6%, 12%, 18% and 24% was maximum at 2
nd
,
3
rd
and 4
th
day of fermentation, accordingly. The
results further indicated that the production of citric
acid was maximum after 6 days of fermentation in
the media containing 24% substrate level with a
mean value of 6.870.12 g 100 mL
-1
. It was found
Int. J. Agric. Appl. Sci. Vol. 5, No.1, 2013
12

that by increasing substrate level, the stationary
phase for citric acid productivity was also delayed
and the optimum recovery of citric acid (59.64%,
5.91 g 100 mL
-1
) with respect to initial total sugar
contents) was achieved in 18% substrate level after
the completion of fermentation period of 6 days
(Table 3). The per day productivity of citric acid also
indicated that under these conditions, the maximum
productivity was achieved after 4 to 5 days of
fermentation (Fig. 9 7).

DISCUSSION

The total sugar contents gradually decreased from
8.28 g 100 mL
-1
to 1.26 g 100 mL
-1
with the
fermentation time up to 8 days. Maximum utilization
of total sugars from sugarcane molasses during 0 to
8 days of fermentation was 7.02 g 100 mL
-1
.

However, on the whole 10.41 g 100 mL
-1
(78.21%)
total sugars were utilized during the course of the
study. The results were in line with the findings of
Jianlong et al. (2000) who reported up to 94.8%
utilization of sugars during citric acid fermentation.
The results were also agreed with Ali et al. (2002)
who observed 18.5 to 96.55 g L
-1
(1.85 to 9.66 g 100
mL
-1
) utilization of sugars during fermentation period
of 6 days.Moreover, the results are closely related to
Haq et al. (2004), who reported 71.33% utilization of
sugars during a fermentation period of 7 days.


2.10 D
4.10 C
5.80 B
6.50 A
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
6 12 18 24
U
t
i
l
i
z
a
t
i
o
n

o
f

t
o
t
a
l

s
u
g
a
r
s

(
g

1
0
0

m
L
-
1
)

Substrate level (%)

Fig. 3: Effect of substrate level on utilization of total sugars during citric acid production from sugarcane
molasses


The optimum conditions observed for citric acid
production were 6 days of fermentation with 24%
substrate level from sugarcane molasses as carbon
substrate for Aspergillus niger. The maximum citric
acid yield from sugarcane molasses was 6.870.12 g
100 mL
-1
(68.7 g L
-1
) with a mean recovery of 51.62%.
However, the maximum citric acid recovery with
respect to initial total sugar contents of the medium
was achieved after 6 days of fermentation in the
media containing 18% substrate level with a mean
recovery of 59.64%. These results were supported by
the findings of Ali et al. (2002) who reported
maximum citric acid production after 6 days of
fermentation from 15% sugar molasses by
Aspergillus niger. They obtained 99.56 3.5 g L
-1
of
citric acid from 150 g total initial sugars with a
recovery of about 66%. The similar results were

U. Farooq, F.M. Anjum, T. Zahoor, S. Rahman, Z. Hayat, K. Akram and E. Ashraf
13

Table 2: Effect of fermentation time and substrate level on utilization of total sugars (g 100 mL
-1
) during citric
acid production from sugarcane molasses
Fermentation time
(days)
Substrate level (%)
Mean
6 12 18 24
0 0.000.00 o 0.000.00 o 0.000.00 o 0.000.00 o 0.000.00 H
1 0.810.04 n 0.840.03 n 0.780.03 n 0.950.03 n 0.850.05 G
2 2.280.09 m 2.440.03 k-m 2.360.02 lm 2.550.04 jl 2.410.15 F
3 2.440.09 k-m 5.070.05 i 5.240.04 i 5.460.04 h 4.560.32 E
4 2.580.10 jk 5.480.04 h 8.130.08 f 8.530.08 e 6.180.49 D
5 2.640.12 j 5.650.02 gh 8.640.07 e 10.020.04 c 6.740.56 C
6 2.680.10 j 5.740.05 g 8.920.06 d 10.210.02 b 6.890.57 B
7 2.720.05 j 5.800.05 g 9.050.08 d 10.340.03 ab 6.980.58 AB
8 2.740.07 j 5.840.04 g 9.100.06 d 10.410.07 a 7.020.58 A
Mean 2.100.11 D 4.100.25 C 5.800.40 B 6.500.46 A


0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
0 1 2 3 4 5 6 7 8
U
t
i
l
i
z
a
t
i
o
n

o
f

t
o
t
a
l

s
u
g
a
r
s

(
g

1
0
0

m
L
-
1
)
Fermentation time (days)
6% Substrate 12% Substrate 18% Substrate 24% Substrate
Fig. 4: Effect of fermentation time and substrate level on per day utilization of total sugars during citric acid
production from sugarcane molasses

reported by Ali et al. (2002) who investigated the
kinetics of submerged citric acid fermentation by
Aspergillus niger using blackstrap molasses as the
basal fermentation media and found that 28C was
the optimum temperature for fermentation.
Similarly, the results also matched with Alben and
Erkmen (2004) and Jianlong et al. (2000) who got
21.9% and 82.2% citric acid yield, from different
substrates.

Haq et al. (2004) reported 31.1 to 96.1 g L
-1
citric
acid production from 15% (w/v) sugarcane molasses
after 7 days of fermentation with 20.7 to 64.0%
recovery. They also observed that 28C temperature
and initial pH 6.0 were the optimum conditions for
fermentation. The results were also agreed to the
findings of El-Holi and Al-Dalaimy (2003) who
observed 106.5 g L
-1
(10.65 g 100 mL
-1
) citric acid
Int. J. Agric. Appl. Sci. Vol. 5, No.1, 2013
14




0.00 F
0.54 E
1.55 D
2.98 C
4.09 B
4.41 A
4.47 A 4.45 A 4.44 A
0.00
1.00
2.00
3.00
4.00
5.00
0 1 2 3 4 5 6 7 8
C
i
t
r
i
c

a
c
i
d

(
g

1
0
0

m
L
-
1
)
Fermentation time (Days)

Fig. 5: Effect of fermentation time on citric acid production from sugarcane molasses


1.20 D
2.58 C
3.84 B
4.35 A
0.00
1.00
2.00
3.00
4.00
5.00
6 12 18 24
C
i
t
r
i
c

a
c
i
d

(
g

1
0
0

m
L
-
1
)
Substrate level (%)

Fig. 6: Effect of substrate level on citric acid production from sugarcane molasses

production from whey supplemented with 15%
sucrose after a fermentation period of 16 days by
Aspergillus niger. The difference in fermentation
time and citric acid production might be due to more
initial total sugar contents in the fermentation
media. Due to higher initial level of total sugars, the
time of completion of fermentation was also
increased and ultimately more citric acid production
was obtained. The results were supported by the
findings of Watanabe et al. (1998), who got 779 g L
-1
U. Farooq, F.M. Anjum, T. Zahoor, S. Rahman, Z. Hayat, K. Akram and E. Ashraf
15

and 987 g L
-1
and 922 g L
-1
and 1023 g L
-1
citric acid
production within 3 to 9 days of fermentation from
the concentrated hydrolysate containing 150 g L
-1
of
reducing sugars. They achieved maximally 682%
yield of citric acid based on the reducing sugars
supplied.


Table 3: Effect of fermentation time and substrate level on citric acid production (g 100 mL
-1
) from sugarcane
molasses

Fermentation
Time (days)
Substrate level (%)
Mean
6 12 18 24
0 0.000.00 h 0.000.00 h 0.000.00 h 0.000.00 h 0.000.00 F
1 0.480.02 g 0.540.02 g 0.510.02 g 0.630.02 g 0.540.03 E
2 1.370.02 f 1.550.04 f 1.560.03 f 1.720.05 f 1.550.09 D
3 1.460.03 f 3.270.10 e 3.490.10 de 3.670.04 d 2.980.22 C
4 1.530.01f 3.550.09 de 5.510.15 c 5.770.10 bc 4.090.34 B
5 1.510.02 f 3.600.08 de 5.780.12 bc 6.750.18 a 4.410.38 A
6 1.500.03 f 3.590.08 de 5.910.15 b 6.870.12 a 4.470.39 A
7 1.480.03 f 3.580.09 de 5.890.16 b 6.860.16 a 4.450.39 A
8 1.470.03 f 3.570.08 de 5.880.16 b 6.840.20 a 4.440.39 A
Mean 1.200.06D 2.580.16 C 3.840.27 B 4.350.31 A
Means carrying same letters are not significantly different


0.00
0.50
1.00
1.50
2.00
2.50
0 1 2 3 4 5 6 7 8
C
i
t
r
i
c

a
c
i
d

(
g

1
0
0

m
L
-
1
)
Fermentation time (days)
6% Substrate 12% Substrate 18% Substrate 24% Substrate

Fig. 7: Effect of fermentation time and substrate level on per day citric acid production from sugarcane molasses


CONCLUSION

The optimum citric acid production was achieved
after 6 days of fermentation in 24% substrate level
using Aspergillus niger as a culture. The maximum
citric acid yield was 6.870.12 g 100 mL
-1
(68.7 g L
-1
)
with a mean recovery of 51.62% with respect to
initial total sugar contents (13.310.15 g 100 mL
-1
) of
the media. Although, the maximum citric acid was
produced after 6 days of fermentation in the media
containing 24% substrate level but the recovery of
citric acid was higher after 6 days of fermentation in
the media containing 18% substrate level. The citric
acid production on 6
th
day of fermentation at 18%
substrate level was 5.91 g 100 mL
-1
with a recovery
of 59.64% with respect to initial total sugar contents
(9.910.20 g 100 mL
-1
). Therefore, it was concluded
that the 6 days of fermentation and 18% substrate
Int. J. Agric. Appl. Sci. Vol. 5, No.1, 2013
16

level were the optimum conditions for citric acid
recovery from sugarcane molasses.

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