Process Biochemistry 38 (2003) 1731
/1738

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Citric acid production by solid state fermentation using sugarcane

bagasse
D. Kumar a, V.K. Jain b, G. Shanker b, A. Srivastava a,*
a
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, New Delhi 110016, India
b
Department of Biochemical Engineering and Food Technology, H.B. Technological Institute, Kanpur 2, India

Received 30 April 2002; received in revised form 7 May 2002; accepted 30 August 2002

Abstract

A solid state fermentation (SSF) method was used to produce citric acid by Aspergillus niger DS 1 using sugarcane bagasse as a
carrier and sucrose or molasses based medium as a moistening agent. Initially bagasse and wheat bran were compared as carrier.
Bagasse was the most suitable carrier, as it did not show agglomeration after moistening with medium, resulting in better heat and
mass transfer during fermentation and higher product yield. Different parameters such as moisture content, particle size, sugar level
and methanol concentration of the medium were optimised and 75% moisture level, 31.8 g sugar/100 g dry solid, 4% (v/w) methanol
and particles of the size between 1.2 and 1.6 mm were found to be optimal. Sucrose and clarified and non-clarified molasses medium
were also tested as moistening agents for SSF and under optimised conditions, 20.2, 19.8 and 17.9 g citric acid /100 g of dry solid
with yield of 69.6, 64.5 and 62.4% (based on sugar consumed) was obtained in sucrose, clarified and non-clarified molasses medium
respectively, after 9 days of fermentation.
# 2002 Published by Elsevier Science Ltd.

Keywords: Citric acid production; Solid state fermentation; Aspergillus niger

1. Introduction production because of the presence of metal ions is

Citric acid is a commercially valuable microbial
product produced mainly by submerged fungal fermen-
tation of sucrose or molasses medium using Aspergillus
niger [1,2]. Laxminarayan et al. [3] and Chaudhary et al.
[4] have used solid state fermentation (SSF) in order to
increase the efficiency of citric acid production and in
the recent years there has been increasing interest in the
use of SSF process as an alternative to submerged
fermentation [5,6]. This is because SSF has a lower
energy requirement, higher product yield with little risk
of bacterial contamination, generates less wastewater
and environmental concerns regarding the disposal of
solid waste [7]. The biosynthesis of citric acid by A. niger
is highly sensitive to the concentration of metal ions
(Fe , Mn , Zn, etc.) in submerged or liquid
surface fermentation [1,2]. Inhibition of citric acid
ineffective in SSF [8,9] and Shankaranand and Lonsane
[10] have reported that the addition of these metal ions
or minerals to molasses and wheat bran based medium
increased citric acid production by 1.4
/1.9 times in SSF.
Sugarcane bagasse, wheat bran and molasses are
produced abundantly in India. An agricultural residue,
bagasse is generally used as a fuel. It can also be utilised
as a carrier for SSF [11] and the present work was
undertaken to optimise process conditions, including
particle size, moisture content, sugar level and methanol
concentration of the medium for the production of citric
acid using agricultural residues (bagasse and wheat
bran) and molasses by SSF.
2. Material and methods
2.1. Organism

* Corresponding author. Tel.: /91-11-6596192. A citric acid producing strain of A. niger , isolated and
E-mail address: ars@dbeb.iitd.ernet.in (A. Srivastava). developed in this laboratory as described below was
0032-9592/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 2 5 2 - 2
1732 D. Kumar et al. / Process Biochemistry 38 (2003) 1731
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Fig. 1. Effect of carrier on citric acid production by SSF.
used in this study. It was maintained on potato dextrose
agar (PDA) slants, stored at 4 8C and subcultured every
2 weeks.
2.2. Media preparation
Bagasse was dried in an oven, cut into small pieces,
grounded and screened to collect four fractions of
different particle sizes, less than 0.64 mm, between
0.64 and 1.2 mm, 1.2
/1.6 and 1.6
/2 mm. Bagasse (3
g) of desired particle size was taken in 250 ml Erlen-
meyer flasks and moistened with medium (15
/20%
sucrose or molasses, 0.25%, NH4NO3, 0.1%, KH2PO4,
0.025%, MgSO4 and 0.004%, CuSO4, pH 4.0) to set the
desired moisture level. Media were sterilised at 121 8C
for 60 min to provide proper cooking of the substrate
and to increase its susceptibility to microbial attack.
Wheat bran media were similarly prepared.
Sugarcane molasses was clarified by potassium ferro-
cyanide treatment. Sugarcane molasses was diluted with
double distilled water to the desired concentration and
the pH was adjusted to 4.5. Heavy metal ions were then
precipitated by adding 0.1% (w/v) potassium ferrocya-
nide followed by heating at 90 8C for 20 min. The
precipitated heavy metals were removed by filtration
and filtrate was passed through activated charcoal to
obtain clarified molasses.
2.3. Isolation of citric acid producing organism
Pieces of lemon were placed on soil at different places
and after a few days fungal growth appeared in each
piece of lemon. The spores from each piece were
transferred separately into PDA plates and incubated
at 30 8C. After 2 days growth appeared in each plate.
Spore suspensions were prepared from each plate
separately, diluted suitably, streaked on other PDA
plates and incubated at 30 8C for 2 days. Spores from
single isolated colonies were taken from each plate,
transferred aseptically to PDA slants and incubated at
30 8C for 2 days. In all 400 isolates were obtained and
stored at 4 8C.
2.4. Screening of isolates
Filter paper disks in Petri dishes were sterilised and
soaked with sterilised sucrose based medium containing
universal indicator solution of the pH range 1
/10.5.
Petri dishes were inoculated with spores of different
isolates and incubated at 30 8C for 3 days. Acid
producing strains were detected by the formation of
coloured (pink) zone around the colony. Zone diameter
was taken as a criterion of good acid producing ability
of isolates. Of these acid producers, citric acid producing
strains were identified by cultivating on bagasse mois-
tened with sucrose based medium in 250 ml flasks for 8
days. Fermented material was extracted using distilled
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Fig. 2. Effect of particle size on citric acid production in SSF. Symbols sp1, sp2, sp3, and sp4 are representing sugar concentrations while p1, p2, p3,
and p4 represent citric acid concentrations at four different particle sizes.
water and citric acid was estimated in extracts. Of these
isolates, A. niger DS 1 was found to be the best citric
acid producer and therefore selected for further work.
2.5. Inoculum
One milliliter spore suspension of the concentration
2 /107 spores/ml was used as a inoculum in each
experiment.
2.6. Solid state fermentation
Each flask, containing medium was inoculated with 1
ml of spore suspension followed by mixing and incuba-
tion at 30 8C, inside the humidity-controlled incubator.
Methanol was added to the medium before inoculation.
One flask was harvested every day for the estimation of
citric acid produced and sugar consumed, till whole
sugar consumed. Sampling was continued until all the
sugar of the medium was consumed.
2.7. Analytical methods
Sugar was estimated by the phenol sulphuric acid
method of Dubois et al. [12] and citric acid by the acetic
anhydride and pyridine method of Marier and Boulet
[13].
1733
3. Results and discussion
The present work consisted of comparison and
selection of suitable carrier (bagasse/wheat bran) for
citric acid production by SSF. The effects of different
parameters including moisture content, particle size,
sugar level and methanol concentration in the medium,
have been studied.
3.1. Comparison and selection of carrier
Initially bagasse and wheat bran were used as a carrier
for citric acid production by SSF at 65% moisture level
and 29.6 g sugar/ 100 g of dry solid. The citric acid
production data are shown in Fig. 1. Citric acid
production and sugar consumption were higher in
bagasse medium than in wheat bran medium. The
maximum citric acid concentration on bagasse and
wheat bran medium was 9.4 and 6.2 g/100 g of dry
solid, respectively after 9 days of fermentation. It was
also observed that wheat bran agglomerated at a
moisture level of 65% whereas bagasse existed in discrete
form at this moisture level. Agglomerations affect uni-
form mixing of the substrate and therefore affect
growth, heat (metabolic heat) transfer, mass (O2 in
take and CO2 evolution) transfer and product forma-
tion. As a result wheat bran medium produced lower
yields than bagasse medium. Bagasse did not show
agglomeration even at 85% moisture level, resulting in
good mixing of substrate, better heat and mass transfer
1734 D. Kumar et al. / Process Biochemistry 38 (2003) 1731
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Table 1
Calculated data on citric acid production at different particle size after 9 days of fermentation

S. No. Particle size (mm) Citric acid (g/100 g) Sugar consumption (%) Yield (%)

1 M1 ( B/0.64) 9.2 97.6 31.8

2 M2 (0.64
/1.2) 10.4 98.6 35.9
3 M3 (1.2
/1.6) 10.9 98.0 37.3
4 M4 (1.6
/2) 8.6 96.0 30.3

Fig. 3. Effect of moisture content on citric acid production in SSF. Symbols SM1, SM2, SM3, and SM4 are representing sugar concentrations, while
m1, m2, m3, and m4 represent citric acid concentrations at 50, 60, 70, and 80% moisture levels, respectively.

Table 2
Calculated data on citric acid production at different moisture level after 9 days of fermentation

S. No. Moisture content (%) Citric acid (g/100 g) Sugar consumption (%) Yield (%)

1 55 8.8 96.6 30.9

2 65 10.7 98.0 36.9
3 75 11.2 98.6 38.4
4 85 10.5 98.6 36.0

and higher citric acid production. Bagasse was therefore
a superior carrier to wheat bran and was selected for
further experiments.
3.2. Effect of particle size
The bagasse of four different particle sizes was used to
prepare four types of medium viz. M1 (particles B/0.64),
M2 (particles between 0.64 and 1.2 mm), M3 (particles
between 1.2 and 1.6 mm) M4 (particles between 1.6 and
2.0 mm). Fermentation was carried out at a moisture
level of 65% and sugar level 29.6 g/100 g of dry solid, for
the production of citric acid. Results are shown in Fig. 2
and some calculated data are given in Table 1. The
maximum concentration of citric acid in medium M1,
M2, M3 and M4 was 9.2, 10.4, 10.9 and 8.6 g/100 g of dry
solid, after 9 days of fermentation. The maximum citric
acid production was obtained from media M3. The rate
of citric acid production was also maximal in M3 media.
The lowest citric acid production was obtained from M4.
 D. Kumar et al. / Process Biochemistry 38 (2003) 1731
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Fig. 4. Effect of initial sugar level on citric acid production in SSF. Symbols M1, M2, M3 and M4 are representing four different medium containing
initial sugar concentrations 26.5, 29.6, 31.8, and 35 g/l while CM1, CM2, CM3 and CM4 are representing citric acid production in these media M1,
M2, M3 and M4, respectively.

Table 3
Calculated data on citric acid production at different sugar level after 9 days of fermentation

S. No. Initial sugar (g/100 g) Citric acid (g/100 g) Sugar consumption (%) Yield (%)

1 M1 (26.5) 9.4 98.5 36.1

2 M2 (29.6) 11.2 98.0 37.9
3 M3 (31.8) 12.1 97.5 38.8
4 M4 (35.2) 11.7 90.8 36.5

Citric acid production in media M2 was very close to M3
whereas in M1 citric acid production was less. This is
presumably due to medium containing larger particles
(M3) exhibiting high porosity and hence resulting in
better heat and mass transfer, which increased citric acid
production. However, very large particles of the medium
(M4) reduced substrate availability to the microbe,
resulting in lower citric acid production. In the case of
smaller particles of the medium (M1), heat and mass
transfer was poor resulting in poor citric acid produc-
tion. The optimum particle size of the medium was M3
which was selected for further studies.
3.3. Effect of moisture content
Experiments were carried out at four different moist-
ure levels viz. 55, 65, 75 and 85%, sugar level 29.6 g /100
g of dry solid and particle size 1.2
/1.6 mm. Fig. 3 shows
the effect of moisture content on citric acid production
by SSF. Some calculated data are give in Table 2. The
maximum citric acid concentration 8.8, 10.7, 11.2 and
10.5 g/100 g of dry solid was obtained from medium
containing 55, 65, 75 and 85% moisture, respectively,
after 9 days of fermentation. Citric acid production and
sugar consumption varied considerably with the moist-
ure content of the medium. Maximum citric acid
production was obtained at 75% moisture level. Both
citric acid production and sugar consumption were
lowest at 55% moisture level. Sugar consumption rate
increased considerably with moisture level, up to 75%.
Sugar consumption was similar at 75 and 85% moisture
levels. These results indicate that the higher moisture
level (85%), probably increased substrate availability to
the fungus but reduced the porosity of the medium and
hence the heat and mass transfer. Increased sugar
consumption may therefore be due to increase in
substrate availability and reduced citric acid may be
due to poor heat and mass transfer and poor oxygen
1736 D. Kumar et al. / Process Biochemistry 38 (2003) 1731
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Fig. 5. Effect of methanol on citric acid production in SSF. Symbols s1, s2, s3, s4, and s5 are representing sugar concentrations at 0, 2, 3, 4, and 5%
methanol, respectively in the medium while C, M1, M2, M3, M4 represents citric acid concentrations in presence of 0, 2, 3, 4 and 5% methanol in
medium.

Table 4
Calculated data on citric acid production at different methanol concentration after 9 days of fermentation

S. No. Methanol conc. (%) Citric acid (g/100 g) Sugar consumption (%) Yield (%)

1 Control 12.2 97.8 39.2

2 2 14.2 95.9 46.5
3 3 19.2 94.1 64.2
4 4 20.6 93.1 69.6
5 5 14.4 86.2 52.6

availability. Lower moisture (55%) also reduced sub-
strate availability to the fungus resulting in lower citric
acid production and sugar consumption. The maximum
38.4% citric acid yield was obtained at 75% moisture
level, which was optimal and selected for further
experiments.
3.4. Optimisation of sugar level of the medium
Four different media viz. M1, M2, M3 and M4 with
sugar levels of 26.5, 29.6, 31.8 and 35.0 g/100 g of dry
solid, respectively, were used for citric acid production
by SSF at a 75% moisture level. The results are shown in
Fig. 4 and some calculated data on citric acid produc-
tion are given in Table 3. In M4 medium both citric acid
production and sugar consumption were decreased
whereas in M1 and M2 sugar consumption was similar
but citric acid production was slightly increased in M2.
The maximum citric acid concentration 12.1 g /100 g of
dry solid was obtained in medium M3 with a yield of
38.8% and sugar consumption of 97.5%. Therefore this
sugar level was selected.
3.5. Effect of methanol
Media containing 2, 3, 4 and 5% (v/w) methanol along
with a control, at a sugar level of 31.8 g/100 g of dry
solid and a moisture level of 75% were used to see the
effect of methanol on citric acid production by SSF.
Results are shown in Fig. 5. Some calculated data are
given in Table 4. Citric acid production increased with
increasing methanol concentration in the medium up to
4%. At methanol concentration higher than 4%, citric
acid production was reduced. The maximum concentra-
tion of citric acid, 20.6 g/100 g of dry solid, with yield of
69.6% and sugar consumption of 94.1% was obtained
after 9 days of fermentation. Methanol reduced growth
of the fungus, sporulation and sugar consumption but
 D. Kumar et al. / Process Biochemistry 38 (2003) 1731
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Fig. 6. Citric acid production from clarified and non-clarified molasses. Symbols s1, s2, ps1 and ps2 represent sugar concentrations. CM and NCM
are representing citric acid production in clarified and non-clarified molasses medium in absence of methanol while PCM and PNCM are exhibiting
citric acid concentration in clarified and non-clarified molasses medium in presence of methanol.

Table 5
Calculated data on citric acid production from clarified and non-clarified molasses after 9 days of fermentation

S. No. Molasses medium Citric acid (g/100 g) Sugar consumption (%) Yield (%)

1 Clarified molasses (without methanol) 11.2 98.1 35.9

2 Non-clarified molasses (without methanol) 10.2 97.5 32.9
3 Clarified molasses (with 4% methanol) 19.8 95.6 64.5
4 Non-clarified molasses (with 4% methanol) 17.8 93.4 60.3

increased citric acid yield by 1.8-fold. The increase in
citric acid yield with methanol is a general phenomenon
and is commonly used in citric acid production [1]. Hang
and Woodams [14
/16] used 3% methanol and obtained
similar stimulatory effect on citric acid production by
SSF using A. niger NRRL 567. The mechanism of
increase in citric acid yield in the presence of methanol is
not known. Dasgupta et al. [17] and Hamissa [18] have
reported that moderate concentrations (1
/3%) of
methanol decreased the iron and manganese uptake by
the fungus and doubled the citric acid yield. Maddox et
al. [19] reported that methanol increase the permeability
of cells to citrate.
3.6. Citric acid production using clarified and non-
clarified molasses by SSF
Being a cheaper carbon source, molasses is preferred
for citric acid production. As reported by several
authors [1,4], the presence of metal ions in molasses
enhances growth of the fungus but inhibits citric acid
production and hence the clarification of molasses was
required. A SSF was carried out using clarified and non-
clarified molasses, in the presence and absence of
methanol (to examine the sensitivity of fungus to metal
ions for citric acid production in SSF) at an initial sugar
level of 31.8 g/100 g of dry solid and a moisture level
75%. The results are shown in Fig. 6. In the absence of
methanol, the maximum concentration of citric acid,
11.2 and 10.2 g/100 g of dry solid with a yield of 35.9
and 32.9. In the presence of methanol the maximum
concentration of citric acid 19.8 and 17.9 g/100 g of dry
solid with yield of 64.5 and 60.3% (Table 5) was
obtained from clarified and non-clarified molasses in
SSF after 9 days of fermentation. The rate of sugar
consumption was slightly higher in non-clarified mo-
lasses medium than clarified molasses medium. This
indicates that the presence of metal ions in molasses
enhanced growth of the fungus and reduced citric acid
production. The adverse effect of metal ions (present in
1738 D. Kumar et al. / Process Biochemistry 38 (2003) 1731
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molasses) on citric acid production was not very
effective as the yields of citric acid, which were similar.
This may be due to the unavailability of free flowing
water in SSF system, transport of these metal ions from
medium to the fungal cells being difficult. It is also
possible that metal ions might be adsorbed on bagasse
particles and therefore less available to the microbe.
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