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CA production by Koji fermentation using banana peel as a novel substrate

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 Bioresource Technology 101 (2010) 5552–5556

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Citric acid production by Koji fermentation using banana peel as a novel substrate
Alagarsamy Karthikeyan a, Nallusamy Sivakumar b,c,*
a
Department of Biotechnology, Alagappa University, Karaikudi, Tamil Nadu 630 003, India
b
Department of Biology, Sultan Qaboos University, P.O. Box 36, PC 123, Muscat, Oman
c
Department of Microbiology, J.J. College of Arts and Science, Pudukkottai, Tamil Nadu, India

a r t i c l e i n f o a b s t r a c t

Article history: The growing demand for citric acid and the current need for alternative sources have encouraged biotech-
Received 4 August 2009 nologists to search for novel and economical substrates. Koji fermentation was conducted using the peels
Received in revised form 10 January 2010 of banana (Musa acuminata) as an inexpensive substrate for the production of citric acid using Aspergillus
Accepted 18 February 2010
niger. Various crucial parameters that affect citric acid production such as moisture content, temperature,
Available online 9 March 2010
pH, inoculum level and incubation time were quantified. Moisture (70%), 28 °C temperature, an initial pH
3, 108 spores/ml as inoculum and 72 h incubation was found to be suitable for maximum citric acid pro-
Keywords:
duction by A. niger using banana peel as a substrate.
Koji fermentation
Banana peel
Ó 2010 Elsevier Ltd. All rights reserved.
Citric acid
Aspergillus niger

1. Introduction available at a cheap price, in all seasons, throughout the country.

Production of citric acid by Aspergillus niger is one of the most
commercially utilized examples of fungal overflow metabolism.
Citric acid is extensively used in dairy, food, beverage, pharmaceu-
tical and biochemical industries. Currently the annual production
of citric acid is around 1.6 million tons (Sauer et al., 2008). The
search for inexpensive substrates is necessary to reduce the pro-
duction cost of citric acid. Considerable interest has been shown
in using agricultural wastes for citric acid production because of
the problems in waste management faced by agro-based indus-
tries, particularly in developing countries. The current emphasis
is on biological conversion of agricultural wastes into value-added
products. For this purpose, different agro-industrial residues such
as grape pomace, apple pomace, banana, sugar beet, okara, jack
fruit carpel and kiwi fruit peel were investigated as possible sub-
strates (Angumeenal and Venkappayya, 2005; Hang and Woodams,
1995; Khare et al., 1995; Vandenberghe et al., 2000; Sassi et al.,
1991; Shojaosadati and Babeipour, 2002). In this study, we have ta-
ken peels of banana (Musa acuminata) as a novel substrate for the
production of citric acid through Koji fermentation using A. niger.
Banana is one of the most abundantly available fruits in tropical
countries. World production of banana is estimated at 48.9 million
tones out of which 10.4 million tones, is contributed by India. India
is the leading country in the production of banana followed by Bra-
zil, Indonesia, Philippines, China and Australia. In India, peels are
* Corresponding author. Address: Department of Biology, Sultan Qaboos Univer-
sity, P.O. Box 36, PC 123, Muscat, Oman. Tel.: +968 24141496; fax: +968 24141437.
E-mail addresses: apnsiva@squ.edu.om, apnsiva@yahoo.com (N. Sivakumar).
All parts of banana tree are useful to human beings except the peel.
In some villages in India, banana peel is seldom used as cattle feed
and it is discarded simply as garbage. Banana peel is an organic
waste that is rich in carbohydrate and other nutrients. Hence the
banana peel was tried in this study as a cheap substrate for citric
acid production.
2. Methods
2.1. Collection of banana peels
Fresh banana was procured in the local market, Pudukkottai,
TamilNadu, India. The ripened fresh banana peels without any
damage were carefully taken and stored at 4 °C. The stored peels
were used for the experiment within 24 h.
2.2. Analysis of banana peel
Moisture content was determined in separate portions of the
freshly removed peels. The sample was then dried at 80 °C and
ground for analysis. Triplicate samples of dried banana peel were
analyzed for carbohydrate, crude protein, crude fat, crude fiber
and total ash contents following the methods of Clarke et al.
(2008), Essien et al. (2005) and Horwitz (1980).
2.3. Organism
Aspergillus niger MTCC 282 was obtained from the Microbial
Type Culture Collection, Institute of Microbial Technology, Chandi-

0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.02.063
 A. Karthikeyan, N. Sivakumar / Bioresource Technology 101 (2010) 5552–5556
garh, India. The organism was maintained on Czapeck Dox agar
slants at 4 °C and renewed once in a month.
2.4. Inoculum
Aspergillus niger spores for inoculum were produced in 50 ml of
Czapek Dox medium in a 250 ml Erlenmeyer flask and incubated at
28 °C for 8 days. A spore suspension was prepared by adding 50 ml
distilled water with Tween 80 (2%) and was stored at 4 °C for a
maximum of 3 weeks. The suspension contained 1010 spores/ml.
2.5. Koji fermentation
A simple Koji fermentation process was followed which is quite
similar to solid-state fermentation. The collected fresh peels were
washed twice with sterile distilled water to remove adhering dust.
Then their finger stalk and black base regions were removed.
Thereafter, one kg of peels was steamed with one liter of water
in order to get the desired moisture content. The steamed peels
were homogenized for five minutes under aseptic conditions. After
homogenization, the pH of the steamed peels was 5.12. It was ad-
justed to different pH ranges using 1 N HCl/NaOH. From the pasty
fibrous material, 2 cm thick Koji beds were prepared in a clean
sterilized glass trough having 30 cm diameter. The glass trough
was used as a fermentation vessel. After inoculation with A. niger
the Koji bed was covered with cheesecloth. The cloth cover allowed
adequate gas exchange. The troughs were placed in a sterile cham-
ber and fermented at different experimental conditions. Citric acid
was measured by a spectrophotometric method using pyridine and
acetic anhydride reagents (Sakurai et al., 1997).
2.6. Determination of fermentation factors
Factors such as moisture content, temperature, pH, inoculum le-
vel and incubation time affecting the citric acid production by A. ni-
ger were standardized by adopting the search technique by varying
one factor at a time. The defined parameter of one experiment was
followed for succeeding experiments.
2.6.1. Effect of moisture content
To study the effect of moisture content on citric acid produc-
tion, different sets of banana peels were steamed with water for
different periods to get different moisture content ranging from
50–90%. A portion of steamed material was dried at 105 °C for a
constant weight. The weight of the dried material was recorded
and the percentage of moisture was calculated (Ellaiah et al.,
2002). The moisture content favoring maximum citric acid produc-
tion was followed for subsequent experiments.
2.6.2. Effect of incubation temperature
To find out the influence of temperature, the citric acid fermen-
tation was carried out at 26, 28, 30, 32 and 34 °C. The temperature
giving high amount of citric acid was taken as an optimum temper-
ature. The optimum temperature for citric acid production derived
from this experiment was applied for subsequent evaluation.
2.6.3. Effect of pH
The effect of initial pH of the substrate was also evaluated by
conducting experiments with initial pH of 2, 3, 4, 5, 6 and 7. The
other parameters were kept at their optimum levels.
2.6.4. Effect of inoculum level
To define an appropriate level of inoculum for citric acid pro-
duction, A. niger spores at various spore concentration levels such
as 104, 106, 108, 1010 and 1012/ml were used. The inoculum level
5553
giving maximum citric acid production was taken as an optimum
level for fermentation.
2.6.5. Effect of incubation period
Experiments were conducted to find out the effect of incubation
period by conducting fermentation for 1–5 days. Optimum levels
of all the other derived parameters were used. The incubation time
giving maximum citric acid production was determined as an opti-
mum incubation time.
2.7. Statistical analysis
The mean values and standard deviations were calculated from
the data obtained from three different experiments. Analysis of
variance was performed by one way ANOVA procedures followed
by Tukey HSD Post Hoc tests using SPSS 11.5. Statistical difference
at p < 0.05 were considered to be significant. Coefficient of deter-
mination (R square) was derived to determine the relationship be-
tween two variables using SPSS 11.5.
3. Results and discussion
Banana peels are rich in nutrients containing 60.2% carbohy-
drates, 8.1% protein, 12.1% fat and 8.2% fiber (Table 1). Because of
the high nutrient content of banana peels it was selected as a pos-
sible alternative substrate for the production of citric acid using A.
niger.
Solid-state fermentation is a well adopted process for cultivat-
ing fungi on natural vegetal resources which are broken down by
excreted hydrolytic enzymes. It offers numerous advantages for
production of bulk chemicals and enzymes. This is because solid-
state processes have lower energy requirements and produce
much less waste water and environmental concerns linked to dis-
posal of solid wastes. Solid-state fermentation is drastically af-
fected by various factors such as selection of a suitable strain,
substrate and process parameters (Pandey et al., 2001). In this
study, moisture content, temperature, pH, inoculum level and
incubation time were varied one at a time to determine the correct
combination of factors to obtain maximum yields of citric acid.
3.1. Effect of initial moisture content
The maximum level of citric acid production was observed at
70% moisture content. A significant increase (p < 0.05) was ob-
served in citric acid production from 50–70%, but this decreased
after that (Fig. 1). Maximum fungal biomass was obtained at the
range of 70–80% initial moisture content of solid-state fermenta-
tion. It was already reported that at 78% moisture had increased
the citric acid production (Shojaosadati and Babeipour, 2002).
Moisture content of solid-state fermentation interferes with the
physical properties of the solid particle and affects the process. In-
creased moisture reduces the porosity of the substrate (Kumar
et al., 2003) and thereby limiting the oxygen transfer (Pandey
et al., 2000). Low substrate moisture results in low product forma-
tion due to decrease in diffusion of solutes, nutrients and gas to the
Table 1
Composition of banana peel (% dry matter, DM).
Parameters % Dry matter
Dry matter 14.3
Crude protein 8.1
Crude fat 12.1
Crude fiber 8.2
Carbohydrate 60.2
Moisture 78.9
5554 A. Karthikeyan, N. Sivakumar / Bioresource Technology 101 (2010) 5552–5556

d 180 d 20
180 c 20
b b 170 c
170 19 bc

Citric acid (g/kg d.wt)

18

Biomass (g/kg d.wt)

18
Citric acid (g/kg d.wt)

160

Biomass (g/kg d.wt)

160 b
a b 17
150 150 d 16
b 16 a
140 15 140 c
a 130 bc 14
130 a 14 b
120
a 13 120
12 a 12
110 Citric acid Biomass 110 Citric acid Biomass
11
100 10 100 10
50% 60% 70% 80% 90% 26˚C 28˚C 30˚C 32˚C 34˚C
Moisture content Temperature

Fig. 1. Effect of moisture content on citric acid and biomass production by A. niger Fig. 2. Effect of temperature on citric acid and biomass production by A. niger using
using banana peel as a substrate under Koji fermentation (values with the same banana peel as a substrate under Koji fermentation (values with the same alphabet
alphabet are not significantly different at the 0.05 level). are not significantly different at the 0.05 level).

cell during fermentation (Nagadi and Correria, 1992; Ellaiah et al.,

2004). The linear regression in relation to citric acid production citric = 30.89 + 7.98 * biomass
and biomass at different moisture contents is illustrated in R-Square = 0.92
170.00
Fig. 1a (R2 = 0.62).

3.2. Effect of incubation temperature

160.00
Significant variation (p < 0.05) of citric acid production was ob-
Citric acid (g/kgd.wt)

served at different temperature levels. Maximum production was

observed at 28 °C (Fig. 2). Beyond 28 °C, the production was signif-
icantly reduced. Temperature above 28 °C also reduces the biomass 150.00
production indicating the optimum level of temperature for citric
acid and biomass production for A. niger was 28 °C. Similar findings
were reported earlier (Roukas, 2000). However, it was also re-
140.00
ported that 31.5 °C was the optimum temperature for citric acid
production by A. niger (Walid et al., 2007). Citric acid production
and biomass was highly correlated (R2 = 0.92) at different incuba-
tion temperatures (Fig. 2a).
130.00

3.3. Influence of pH 13.00 14.00 15.00 16.00 17.00 18.00

Biomass (g/kg d.wt)
Production of citric acid was greatly influenced by difference in
Fig. 2a. Relationship between citric acid production and biomass at different
initial pH. A significant increase in citric acid production was ob- incubation temperatures.

served in fermentation by increasing the initial pH from 2 to 3.

However, at pH 4, 5, 6 and 7 citric acid production decreased sig-
citric = 89.38 + 4.56 * biomass nificantly (Fig. 3). The pH value maintained at the beginning of fer-
R-Square = 0.62
mentation is important for a specific biomass formation. pH in the
170.00 range of 3–5 was found to be suitable for biomass production be-
cause in this pH range the difference in biomass content was insig-
nificant but significantly higher than the other pH ranges. Different
Citric acid (g/kgd.wt)

180 f 20
160.00
170 19
Citric acid (g/kg d.wt)

Biomass (g/kg d.wt)

18
160 e
17
150 bc bc 16
b
140 15
c ab
130 d ab 14
150.00 a 13
120 b 12
110 Citric acid Biomass a 11
100 10
2 3 4 5 6 7
14.00 15.00 16.00 17.00 18.00 19.00 pH
Biomass (g/kg d.wt)
Fig. 3. Effect of initial pH on citric acid and biomass production by A. niger using
Fig. 1a. Relationship between citric acid production and biomass at different banana peel as a substrate under Koji fermentation (values with the same alphabet
moisture contents. are not significantly different at the 0.05 level).
 A. Karthikeyan, N. Sivakumar / Bioresource Technology 101 (2010) 5552–5556 5555

ranges of optimum pH have been reported for A. niger such as pH 4 180.00

in fermentation with beet molasses and corn steep liquor (Walid
et al., 2007) and pH 2.6 in solid-state fermentation using cassava
bagasse (Prado et al., 2005). Linear regression showed very low
correlation between citric acid production and biomass at different
pH levels (Fig. 3a). citric = 47.34 + 6.44 * biomass
R-Square = 0.59
160.00

Citric acid (g/kgd.wt)

3.4. Influence of inoculum level

The influence of various concentrations of A. niger spores on cit-

ric acid fermentation was quantified. Maximum production was
observed when 108 spores/ml was given as the inoculum (Fig. 4).
Similar findings were reported earlier (Vandenberghe et al., 140.00
2000). However significant variation (p < 0.05) was observed in cit-
ric acid production at various inoculum levels. When inoculum le-
vel lower or higher than the optimum was used for fermentation
process citric acid production decreased. On the other hand, in-
crease in biomass formation was attained by increasing the inocu-
lum concentration. It is imperative to provide an optimum level of 120.00
inoculum for citric acid production because low inoculum may 12.00 14.00 16.00 18.00
give inadequate biomass and reduced citric acid formation Biomass (g/kg d.wt)
whereas high inoculum levels may form excessive biomass and de-
plete the nutrients essential for citric acid production (Sabu et al., Fig. 4a. Relationship between citric acid production and biomass at different
inoculum levels.

e
20
180 c c
c 18
160
Citric acid (g/kg d.wt)

16

Biomass (g/kg d.wt)

140 b
160.00 d 14
c 12
120
100 a 10
b 8
80
citric = 21.26 + 7.07 * biomass 6
60
Citric acid (g/kgd.wt)

4
R-Square = 0.16 40 a Citric acid Biomass
140.00 2
20 0
24 48 72 96 120
Incubation time (h)

Fig. 5. Effect of incubation time on citric acid and biomass production by A. niger
120.00 using banana peel as a substrate under Koji fermentation (values with the same
alphabet are not significantly different at the 0.05 level).

100.00

14.00 15.00 16.00 17.00 18.00 160.00 citric = -77.91 + 13.79 * biomass
Biomass (g/kg d.wt) R-Square = 0.83

Fig. 3a. Relationship between citric acid production and biomass at different initial
pH.
120.00
Citric acid

190 bc c 20
bc
180 b 18
Citric acid (g/kg d.wt)

16
Biomass (g/kg d.wt)

170 80.00
a e
160 14
12
150 d c 10
140 b
8
130 6 40.00
120 4
110 a Citric acid Biomass 2
100 0
4 6 8 10 12
104
10 106
10 108
10 1010
10 1012
10
Inoculum (spores/ml) 7.50 10.00 12.50 15.00 17.50
Biomass
Fig. 4. Influence of inoculum level on citric acid and biomass production by A. niger
using banana peel as a substrate under Koji fermentation (values with the same Fig. 5a. Relationship between citric acid production and biomass at different
alphabet are not significantly different at the 0.05 level). incubation periods.
5556 A. Karthikeyan, N. Sivakumar / Bioresource Technology 101 (2010) 5552–5556
2006). There is a low correlation between citric acid production
and biomass (R2 = 0.59) at various concentrations of initial inocu-
lum levels (Fig. 4a).
3.5. Effect of incubation time
Fermentation was carried out at different incubation periods to
standardize the optimum incubation time. High levels of citric acid
formed at 72 h of incubation. Further incubation results in de-
creased citric acid production (Fig. 5). Maximum biomass produc-
tion was observed at 72 h after that the increase was insignificant.
It has been reported that optimum incubation time for A. niger was
120 h for citric acid production when cassava bagasse was used as
a substrate (Vandenberghe et al., 2000); 4 days in jack fruit carpel
fiber (Angumeenal and Venkappayya, 2005) and 5 days in Kumara
(Lu et al., 1997). Citric acid production and biomass were highly
correlated (R2 = 0.83) at different incubation periods (Fig. 5a).
4. Conclusion
In view of the existing economic recession and escalating cost of
substrates for microbial growth and production, it is necessary to
explore alternative organic substrates for microbial production.
The present study has shown a great potential for utilizing banana
peel as a substrate for the production of citric acid by A. niger. This
is the first attempt to utilize banana peel as a substrate for citric
acid production. Banana peel is an abundant agricultural waste
material that could be used as a cheap alternative medium for
the production of citric acid.
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