Citric Acid Production by Solid State Fermentation Using Sugarcane Bagasse
Citric Acid Production by Solid State Fermentation Using Sugarcane Bagasse
Citric Acid Production by Solid State Fermentation Using Sugarcane Bagasse
www.elsevier.com/locate/procbio
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.
* 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 /1738
used in this study. It was maintained on potato dextrose 2.3. Isolation of citric acid producing organism
agar (PDA) slants, stored at 4 8C and subcultured every
2 weeks. 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
2.2. Media preparation
at 30 8C. After 2 days growth appeared in each plate.
Spore suspensions were prepared from each plate
Bagasse was dried in an oven, cut into small pieces,
separately, diluted suitably, streaked on other PDA
grounded and screened to collect four fractions of plates and incubated at 30 8C for 2 days. Spores from
different particle sizes, less than 0.64 mm, between single isolated colonies were taken from each plate,
0.64 and 1.2 mm, 1.2 /1.6 and 1.6 /2 mm. Bagasse (3 transferred aseptically to PDA slants and incubated at
g) of desired particle size was taken in 250 ml Erlen- 30 8C for 2 days. In all 400 isolates were obtained and
meyer flasks and moistened with medium (15 /20% stored at 4 8C.
sucrose or molasses, 0.25%, NH4NO3, 0.1%, KH2PO4,
0.025%, MgSO4 and 0.004%, CuSO4, pH 4.0) to set the
2.4. Screening of isolates
desired moisture level. Media were sterilised at 121 8C
for 60 min to provide proper cooking of the substrate Filter paper disks in Petri dishes were sterilised and
and to increase its susceptibility to microbial attack. soaked with sterilised sucrose based medium containing
Wheat bran media were similarly prepared. universal indicator solution of the pH range 1 /10.5.
Sugarcane molasses was clarified by potassium ferro- Petri dishes were inoculated with spores of different
cyanide treatment. Sugarcane molasses was diluted with isolates and incubated at 30 8C for 3 days. Acid
double distilled water to the desired concentration and producing strains were detected by the formation of
the pH was adjusted to 4.5. Heavy metal ions were then coloured (pink) zone around the colony. Zone diameter
precipitated by adding 0.1% (w/v) potassium ferrocya- was taken as a criterion of good acid producing ability
nide followed by heating at 90 8C for 20 min. The of isolates. Of these acid producers, citric acid producing
precipitated heavy metals were removed by filtration strains were identified by cultivating on bagasse mois-
and filtrate was passed through activated charcoal to tened with sucrose based medium in 250 ml flasks for 8
obtain clarified molasses. days. Fermented material was extracted using distilled
D. Kumar et al. / Process Biochemistry 38 (2003) 1731 /1738 1733
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 3. Results and discussion
isolates, A. niger DS 1 was found to be the best citric
acid producer and therefore selected for further work. The present work consisted of comparison and
selection of suitable carrier (bagasse/wheat bran) for
citric acid production by SSF. The effects of different
2.5. Inoculum parameters including moisture content, particle size,
sugar level and methanol concentration in the medium,
One milliliter spore suspension of the concentration have been studied.
2 /107 spores/ml was used as a inoculum in each
experiment.
3.1. Comparison and selection of carrier
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 (%)
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 (%)
and higher citric acid production. Bagasse was therefore 2.0 mm). Fermentation was carried out at a moisture
a superior carrier to wheat bran and was selected for level of 65% and sugar level 29.6 g/100 g of dry solid, for
further experiments. the production of citric acid. Results are shown in Fig. 2
and some calculated data are given in Table 1. The
3.2. Effect of particle size 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
The bagasse of four different particle sizes was used to solid, after 9 days of fermentation. The maximum citric
prepare four types of medium viz. M1 (particles B/0.64), acid production was obtained from media M3. The rate
M2 (particles between 0.64 and 1.2 mm), M3 (particles of citric acid production was also maximal in M3 media.
between 1.2 and 1.6 mm) M4 (particles between 1.6 and The lowest citric acid production was obtained from M4.
D. Kumar et al. / Process Biochemistry 38 (2003) 1731 /1738 1735
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 (%)
Citric acid production in media M2 was very close to M3 by SSF. Some calculated data are give in Table 2. The
whereas in M1 citric acid production was less. This is maximum citric acid concentration 8.8, 10.7, 11.2 and
presumably due to medium containing larger particles 10.5 g/100 g of dry solid was obtained from medium
(M3) exhibiting high porosity and hence resulting in containing 55, 65, 75 and 85% moisture, respectively,
better heat and mass transfer, which increased citric acid after 9 days of fermentation. Citric acid production and
production. However, very large particles of the medium sugar consumption varied considerably with the moist-
(M4) reduced substrate availability to the microbe, ure content of the medium. Maximum citric acid
resulting in lower citric acid production. In the case of production was obtained at 75% moisture level. Both
smaller particles of the medium (M1), heat and mass citric acid production and sugar consumption were
transfer was poor resulting in poor citric acid produc- lowest at 55% moisture level. Sugar consumption rate
tion. The optimum particle size of the medium was M3
increased considerably with moisture level, up to 75%.
which was selected for further studies.
Sugar consumption was similar at 75 and 85% moisture
levels. These results indicate that the higher moisture
3.3. Effect of moisture content level (85%), probably increased substrate availability to
the fungus but reduced the porosity of the medium and
Experiments were carried out at four different moist- hence the heat and mass transfer. Increased sugar
ure levels viz. 55, 65, 75 and 85%, sugar level 29.6 g /100 consumption may therefore be due to increase in
g of dry solid and particle size 1.2 /1.6 mm. Fig. 3 shows substrate availability and reduced citric acid may be
the effect of moisture content on citric acid production due to poor heat and mass transfer and poor oxygen
1736 D. Kumar et al. / Process Biochemistry 38 (2003) 1731 /1738
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 (%)
availability. Lower moisture (55%) also reduced sub- dry solid was obtained in medium M3 with a yield of
strate availability to the fungus resulting in lower citric 38.8% and sugar consumption of 97.5%. Therefore this
acid production and sugar consumption. The maximum sugar level was selected.
38.4% citric acid yield was obtained at 75% moisture
level, which was optimal and selected for further
3.5. Effect of methanol
experiments.
Media containing 2, 3, 4 and 5% (v/w) methanol along
3.4. Optimisation of sugar level of the medium 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
Four different media viz. M1, M2, M3 and M4 with effect of methanol on citric acid production by SSF.
sugar levels of 26.5, 29.6, 31.8 and 35.0 g/100 g of dry Results are shown in Fig. 5. Some calculated data are
solid, respectively, were used for citric acid production given in Table 4. Citric acid production increased with
by SSF at a 75% moisture level. The results are shown in increasing methanol concentration in the medium up to
Fig. 4 and some calculated data on citric acid produc- 4%. At methanol concentration higher than 4%, citric
tion are given in Table 3. In M4 medium both citric acid acid production was reduced. The maximum concentra-
production and sugar consumption were decreased tion of citric acid, 20.6 g/100 g of dry solid, with yield of
whereas in M1 and M2 sugar consumption was similar 69.6% and sugar consumption of 94.1% was obtained
but citric acid production was slightly increased in M2. after 9 days of fermentation. Methanol reduced growth
The maximum citric acid concentration 12.1 g /100 g of of the fungus, sporulation and sugar consumption but
D. Kumar et al. / Process Biochemistry 38 (2003) 1731 /1738 1737
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 (%)
increased citric acid yield by 1.8-fold. The increase in enhances growth of the fungus but inhibits citric acid
citric acid yield with methanol is a general phenomenon production and hence the clarification of molasses was
and is commonly used in citric acid production [1]. Hang required. A SSF was carried out using clarified and non-
and Woodams [14 /16] used 3% methanol and obtained clarified molasses, in the presence and absence of
similar stimulatory effect on citric acid production by methanol (to examine the sensitivity of fungus to metal
SSF using A. niger NRRL 567. The mechanism of ions for citric acid production in SSF) at an initial sugar
increase in citric acid yield in the presence of methanol is level of 31.8 g/100 g of dry solid and a moisture level
not known. Dasgupta et al. [17] and Hamissa [18] have 75%. The results are shown in Fig. 6. In the absence of
reported that moderate concentrations (1 /3%) of methanol, the maximum concentration of citric acid,
methanol decreased the iron and manganese uptake by 11.2 and 10.2 g/100 g of dry solid with a yield of 35.9
the fungus and doubled the citric acid yield. Maddox et and 32.9. In the presence of methanol the maximum
al. [19] reported that methanol increase the permeability concentration of citric acid 19.8 and 17.9 g/100 g of dry
of cells to citrate. solid with yield of 64.5 and 60.3% (Table 5) was
obtained from clarified and non-clarified molasses in
3.6. Citric acid production using clarified and non- SSF after 9 days of fermentation. The rate of sugar
clarified molasses by SSF consumption was slightly higher in non-clarified mo-
lasses medium than clarified molasses medium. This
Being a cheaper carbon source, molasses is preferred indicates that the presence of metal ions in molasses
for citric acid production. As reported by several enhanced growth of the fungus and reduced citric acid
authors [1,4], the presence of metal ions in molasses production. The adverse effect of metal ions (present in
1738 D. Kumar et al. / Process Biochemistry 38 (2003) 1731 /1738
molasses) on citric acid production was not very culture of Aspergillus niger on polyurethane foams. Enzyme
Microb Technol 1998;23:149 /56.
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medium to the fungal cells being difficult. It is also support. Biotechnol Lett 1995;17(2):214 /9.
possible that metal ions might be adsorbed on bagasse [10] Shankaranand VS, Lonsane BK. Ability of Aspergillus niger to
particles and therefore less available to the microbe. tolerate metal ions and minerals in SSF system for production of
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