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Bioresource Technology 99 (2008) 69866993

Comparison of pretreatment strategies of sugarcane

baggase: Experimental design for citric acid production
Kianoush Khosravi-Darani *, Alaleh Zoghi
Department of Food Technology Research, National Nutrition and Food Technology Research Institute, Shaheed Beheshti University,
M.C., P.O. Box 19395-4741, Tehran, Iran
Received 7 May 2007; received in revised form 9 January 2008; accepted 10 January 2008
Available online 10 March 2008

Abstract
Solid state fermentation was carried out to compare eciency of acid, alkaline and urea pretreatment of sugarcane bagasse for
production of citric acid using Aspergillus niger ATCC 9142. PlackettBurman statistical design was used to evaluate signicance of variables. Pretreatment of bagasse by urea was known as the most inuential treatment to increase citric acid production (137.6 g/kg of dry
sugarcane bagasse and citric acid yield of 96% based on sugar consumed). Finally, up scaling was achieved to a 20 L solid state fermentor
in which humidity was constant in gas phase and urea-treated sugarcane bagasse. The produced acid concentration and yield in fermentor was 82.38 g/kg of dry substrate and 26.45 g/kg day, respectively.
2008 Elsevier Ltd. All rights reserved.
Keywords: Solid state fermentation (SSF); Citric acid; Pretreatment; Sugarcane baggase; PlackettBurman design (PBD)

1. Introduction
The use of lignocellulosic materials available in agricultural wastes as a source of raw material for citric acid production is of considerable interest because of their
renewable nature and abundance (Amartey et al., 1999).
In the last two decades, a considerable interest has been
shown in using agricultural products and their wastes for
citric acid production by Aspergillus niger (Khosravi-Darani et al., 2008). Among these substrates, sugarcane bagasse
(Luciana et al., 2000; Kumar et al., 2003), wheat bran
(Yamada, 1977) and date waste (Shojaosadati et al.,
1999) have been investigated by Iranian researchers
because of their availability, low cost and high volume of
production. Sugarcane bagasse which is an agricultural residue and generally used as a fuel, consists of water (4652%
w/w); ber (4352% w/w including cellulose 50%, hemicellulose 25% and lignin 25%) and relatively small quantities

Corresponding author. Tel.: +98 21 22376473; fax: +98 21 22360660.

E-mail address: kiankh@yahoo.com (K. Khosravi-Darani).

0960-8524/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2008.01.024

of soluble solids (26% w/w) (Pandey and Soccol, 1998;

Pandey et al., 2000). Thousands of tons of bagasse are discarded daily by the sugarcane processing industries, leading
to a big environmental problem. Thus, there is an urgent
need to nd suitable applications for this waste. One alternative for economic utilization of sugarcane bagasse is its
use as substrate and/or carrier in solid state fermentation
(SSF) processes for production of value added products.
Citric acid is an important commercial product with a
global production more than 1 million tons per year
(Papagianni, 2007). Almost the entire quantity of citric acid
is produced by fermentation, mainly through submerged
fermentation of starch or sucrose based media, using the
lamentous fungus A. niger. The food industry is the largest consumer of citric acid, using almost 70% of the total
production, followed by about 12% by the pharmaceutical
industry and 18% for other applications (Vandenberghe
et al., 1999). There is also an annual growth of 3.54.0%
in demand/consumption rate of citric acid. SSF oers
numerous advantages for the production of bulk chemicals
and enzymes (Vandenberghe et al., 2000). The citric acid
market has been under pressure for more than two years

K. Khosravi-Darani, A. Zoghi / Bioresource Technology 99 (2008) 69866993

and continues to oscillate with prices falling from $2/kg to

$0.70$0.80/kg. Several producers have cut back on production levels or closed down as a result of the adverse
market conditions. Chinese suppliers tend to sell their citric
acid at the lowest possible price in order to bring in hard
currency and this has made it extremely hard for European
suppliers to compete (Soccol et al., 2006). Although the
production by submerged fermentation is still dominating,
but SSF can create new possibilities for producers. Many
by-products and residues of the agro-industry can be used
in the production of citric acid. The use of agro-industrial
residues as support in SSF is economically important and
minimizes environmental problems. Other perspectives
for citric acid production sector are the improvement of
producing strains, which have been carried out by mutagenesis and selection (Soccol et al., 2006).
Pretreatment strategies of lignocellulosic substrates are
necessary for decreasing lag phase of microbial growth
due to increasing availability of carbohydrate bands. Acid
pretreatment has become a state of the art technology for
pretreating any lignocellulosic material (Lee et al., 1999).
It has the advantage of not only solubilizing hemi-cellulose
but also converting solubilized hemi-cellulose to fermentable sugars (Saha and Bothast, 1999). However, depending
on the temperature, the acid pretreatment usually produces
sugar degradation products, such as furfural, which are
inhibitory to the fermentative microorganisms (Saha,
2004). Compared to acid pretreatments, alkaline processes
have less sugar degradation, furan derivative formation is
avoided and many of the caustic salts can be recovered
(Gonzalez et al., 1986). The PlackettBurman design
(PBD) has been frequently used for screening process variables that make the greatest impact on a process (Plackett
and Burman, 1946).
The aim of this study is to compare citric acid production from pretreated (acid, alkaline and urea-treated) and
untreated sugarcane bagasse for citric acid production in
SSF using a culture of A. niger. This work has been undertaken to screen process variables, including nitrogen source
concentration (urea), methanol, steam time, initial sugar
concentration, temperature, time, solvent, pH, inoculum
size, moisture content and spore age for the production
of citric acid using agricultural residue of sugarcane
bagasse and molasse by SSF.
2. Methods
2.1. Microorganism
A. niger ATCC 9142 was obtained from the culture collection, of the Iranian Research Organization for Science
and Technology (IROST). The organism was maintained
on potato dextrose agar (PDA) slants, preserved at 4 C,
and was sub-cultured every month. For inoculum preparation, the cultures were incubated on PDA slants at 30 C
for 4 or 6 days and the spores obtained were suspended
in 8 ml of sterile-distilled water.

6987

2.2. Substrate
Bagasse was dried in an oven and used without grinding
to reduce energy consumption (Gibbons and Westby, 1986)
and facilitate aeration (Shojaosadati and Babaeipour,
2002). Bagasse (3 g) was taken in ask, moistened and supplemented by water and molasses (as describe in Section
2.5) to set the desired moisture and sugar level. Media were
sterilized at 121 C for 30 and 90 min to provide proper
cooking of the substrate and to increase its susceptibility
to microbial attack.
2.3. Pretreatments
Slurry of sugarcane bagasse in 1 N or 5 N HCl (solid
liquid ratio of 10% w/v), was pretreated in a water bath
at 100 C for 1 h. After cooling down it was washed by distilled water and dried in an oven at 100 C. Alkaline pretreatment was achieved by 1 M or 5 M NaOH solution,
(solidliquid ratio of 10% w/v), during 1 h at room temperature. Then it was washed twice with distilled water and
dried in an oven at 100 C. For urea pretreatment bagasse
was soaked with 2% or 4% (w/v) urea solution (solidliquid
ratio of 5% w/v), stored at room temperature for 3 weeks
and dried in an oven at 100 C.
2.4. SSF
Experiments were conducted in 250-ml Erlenmeyer
asks, each containing 3 g of pretreated or crude sugarcane
bagasse, and moistened with the appropriate amount of
distilled water to reach 65% or 75% (v/w) moisture. Substrate was supplemented with sugarcane molasses to contain 14% or 18% (w/w) initial sugar (the water content of
molasses was considered in moisture adjustment). Methanol (as production stimulator) was added at 3% or 4%
(v/w). The pH of the substrate was adjusted to 4 or 5.5 with
NaOH or HCl (low and high value in Table 1). After autoclaving at 121 C for 20 or 90 min, the asks were cooled to
ambient temperature and inoculated with 1 ml of spore suspension containing about 105 or 107 cfu/ml (low and high
values in Table 2). The asks were incubated at 25 C or
30 C in an incubator for 4 or 5 days. Finally a predetermined concentration of nitrogen source (urea) was added
to crude sugarcane bagasse in two levels (2 or 4% w/w),
to evaluate the impact of nitrogen source on citric acid
production.
After the screening of the variables in ask for untreated
and treated sugarcane bagasse, the experiment was scaled
up in a 20 L solid state fermentor (equipped to three trays)
with constant humidity in gas phase. Input of humidity and
aeration to all trays of this bioreactor was distributed
homogeneously. Filling capacity of fermentor was 15 kg
dry bagasse (5 kg for each tray). The fermentor was
equipped to humidier, moisture controller, recorder and
monitor (to show any uctuation of moisture in gas phase

6988

K. Khosravi-Darani, A. Zoghi / Bioresource Technology 99 (2008) 69866993

Table 1
Variables to be monitored in citric acid production from sugarcane
bagassea
Variables

Low level ()

High level (+)

A: Urea concen. (% w/w)

B: Methanol (%v/w)
C: Steam time (min)
D: Initial sugar (% w/w)
E: Temperature (C)
F: Time (day)
G: Solvent
H: pH
I: Inoculum size (cfu/ml)
J: Moisture content (%)
K: Spore age (day)
In acid treatmenta A:
Acid concen. 10% w/v
In urea treatment
Urea concen. %v/w
In alkaline treatment
NaOH concen. 10% w/v

2
3
20
14
25
4
Water
4
105
65
4
1N

4
4
90
18
30
5
Acetone
5.5
107
75
6
5N

2
1M

4
5M

a
All the 11 factors except factor A are the same in untreated, alkaline,
urea and acid treated sugarcane bagasse.

in a long period of passed 6 months) as well as water reservoir to control the humidity in gas phase.
2.5. Extraction of citric acid
At the end of the fermentation period, the fermented
materials were extracted by distilled water with or without
acetone (50%v/v), in two stage on an agitator (30 min in
each stage). After ltration, the ltrates were centrifuged
at 3500g for 10 min, and then supernatant was analyzed
for citric acid and residual sugar.
2.6. Analytical techniques
The pH of the substrate was measured by a pH-meter
equipped with a glass electrode, using a solidliquid ratio
of 10% (w/v) with distilled water. Moisture content of
bagasse was determined by drying the solid at 105 C to
a constant weight (James, 1995). Citric acid was determined spectrophotometrically at 420 nm by the acetic
anhydride-pyridine method (Marier and Boulets, 1958).
Total reducing sugars were measured by dinitrosalicylic
acid method, using glucose as standard (Miller, 1959).
2.7. PlackettBurman design (PBD)
The rst optimization step was to identify the variables
which have signicant eects on citric acid production by
A. niger. The selection of these factors was based on our
prior experience (Fatemi and Shojaosadati, 2001; Khosravi-Darani et al., 2003) and choice of settings reected a wide
but reasonable numerical range (Khosravi-Darani et al.,
2008). Also some changes in the yield were expected for
each factor over the selected range. The important criteria

to choose factor settings for screening design has been mentioned elsewhere (Davies, 1993).
The variables to be evaluated (Table 1) include some
medium components (i.e., moisture content, initial concentration of carbon and nitrogen sources) and environmental
factors (i.e., temperature, pH, time, inoculum size and age,
solvent and time of steam treating). Table 2 also shows
selected experimental variables and a PBD for conducting
12 trials. The elements, + (high level) and  (low level) represent the two dierent levels of the independent variables
examined (Logothetis and Wynn, 1989).
2.8. Range nding
One important factor that aects the performance of
SSF is the moisture content of substrates (Roukas, 1999).
The purpose of this experiment was to determine the suitable moisture level of crude sugarcane bagasse that would
result in the highest citric acid concentration. Decreasing of
the moisture level to the less than 65%, results a sub-optimal product formation due to reduced mass transfer processes, e.g. diusion of solutes and gas to the cell (Ngadi
and Correia, 1992); and increased osmotic pressure (Kargi
et al., 1985). The increase of moisture (more than almost
85%) increases the chance of contamination and decrease
the space between particles, then transformation of oxygen
and carbon dioxide will be reduce (Tran and Mitchell,
1995). So to determine the eect of this factor on response
two levels of 65% and 75% were selected.
Another variable which can inuence the yield and productivity is conditions of seed culture. The suitable condition for induction of ligninolytic activity can be achieved
by selection of suitable inoculum size and age. So, A. niger
ATCC 9142 was grown on PDA slants for 4 and 6 days,
and a spore suspension of about 105 and 107 (cfu/ml)
prepared.
The initial pH of the substrate also aects the performance of sugarcane bagasse fermentation. On the basis
of literature review data range of pH was selected in 4
and 5.5 to determine the eect of this variable on response.
The use of methanol to enhance citric acid production by
A. niger was rst reported by Moyer (1953a,b) whose
results showed that the mould produced the greatest
amount of citric acid from crude sugarcane bagasse in
the presence of methanol at a concentration of 4% (v/w).
Further studies showed that the addition of methanol at
concentrations of 14% (v/v) results in a marked increase
of citric acid formation by A. niger on spent grain liquor
and brewery wastes (Hang et al., 1977; Roukas and
Kotzekidou, 1987) due to increased cell permeability level
and decreased end product repression of related enzyme
(Kapoor et al., 1987; Maddox et al., 1986). To determine
the eect of methanol concentration on yield and productivity two levels of 3% and 4% were selected.
Temperature has a profound inuence on the fungal
production of citric acid from sugarcane bagasse. To determine the eect of temperature on yield and productivity

Each value is the average of two replication.

Four dierent table of PBD has been done with four dierent factor A of Table 1 (acid, urea and alkali concen.) to compare untreated and treated sugarcane.
Factors A through K refer to those in Table 1.
Experimented yield.
Predicted yield.
b

+
+

+
+
+

1
2
3
4
5
6
7
8
9
10
11
12

pred

2.62
7.31
17.37
10.11
27.20
9.66
3.81
3.54
4.95
15.92
2.53
12.69
1.84
6.53
16.84
9.34
26.45
8.87
3.01
2.78
4.18
15.16
6.05
11.92

exp
pred

0.92
0.93
0.70
1.02
0.96
0.91
0.77
0.70
0.75
0.95
0.08
0.97
0.86
0.87
0.80
0.96
0.90
0.85
0.71
0.64
0.69
0.89
0.84
0.91

exp
pred

24.55
26.83
26.87
10.41
13.75
8.51
20.11
15.01
19.93
11.31
11.03
10.59
23.33
25.62
25.65
3.18
12.53
7.27
18.88
13.78
18.72
10.09
9.79
9.34

exp
pred

0.989
0.983
1.011
0.183
0.713
0.351
0.975
0.853
1.017
0.981
0.763
0.919
0.927
0.918
0.944
0.528
0.647
0.286
0.913
0.788
0.955
0.918
0.699
0.855

exp
pred

15.72
9.22
11.32
8.72
14.92
26.08
22.54
4.40
13.98
9.86
14.88
9.68
14.67
8.16
10.28
7.66
13.84
25.04
21.52
7.22
12.91
8.78
13.84
8.63

exp
pred

0.765
0.563
0.595
0.987
0.901
0.977
1.045
0.197
1.009
0.821
0.955
0.867
0.701
0.499
0.531
0.923
0.837
0.913
0.981
0.508
0.946
0.755
0.892
0.804

exp
pred

22.94
14.75
9.32
1.82
5.60
14.04
6.56
5.50
8.74
8.38
12.52
6.32
22.22
14.02
8.59
1.09
4.84
13.32
5.82
2.54
8.03
7.65
11.78
5.58

exp

0.984
0.970
0.764
0.108
0.660
0.618
0.366
0.184
1.024
0.916
0.950
0.792

+
+
+

+
+

+
+

+
+
+

+
+

+
+
+

+
+

+
+
+

+
+

+
+
+

+
+

+
+

+
+

+
+

+
+
+

+
+

7
12
2
10
6
3
11
1
4
5
9
8

prede

+
+
+

0.930
0.917
0.710
0.053
0.605
0.564
0.310
0.422
0.971
0.862
0.896
0.738

expd
Randomized trails
K
J
I
H
G
F
E
D
C
B
Ac
Run No

+
+

+
+
+

Productivity
(g/kg.day)
Urea treated

Yield%
(acid/sugar)
Productivity
(g/kg.day)

Alkaline treated

Yield%
(acid/sugar)
Productivity
(g/kg.day)

Acid treated

Yield%
(acid/sugar)
Productivity
(g/kg.day)

untreated

Yield%
(acid/sugar)

Codded setting for factors

Table 2
Twelve-trial PBD to study eleven factors in citric acid production from sugarcane bagasse: acomparison of experimented and predicted yield and productivity in untreated and pretreated sugarcane bagasseb

K. Khosravi-Darani, A. Zoghi / Bioresource Technology 99 (2008) 69866993

6989

two levels of 25 and 30 C were selected. Time is also a very

important factor in industry, so fermentation duration of 4
and 5 days were selected to determine the relative importance of this factor. Previous studies also reported that initial sugar concentration of 1422% is optimum in industrial
fermentations (Rohr et al., 1983). The initial sugar concentration of sugarcane bagasse was about 2.5% (much lower
than optimum), so sugarcane molasses was added at 14%
or 18% (w/w). The initial sugar requirements for growth
and acid production varies from species to species and
from strain to strain. The importance of this variable on
citric acid production and growth has been reported
(Fatemi and Shojaosadati, 1999, 2001).
Leaching is an important unit operation that extracts
one or more constituents of a solid mixture by contact with
a solvent and the countercurrent multiple contact system is
commonly used to obtain the most concentrated solution
of a product (Hang and Woodams, 1989). Acetone is
known as a more eective solvent for leaching of citric acid
than water, and extraction eciency of 90% can be
achieved by three leaching stages by acetone. Moreover,
acetone can be easily recovered and recycled to reduce
the cost of operation due to its lower boiling point
(56.2 C).
Also it has been established that the accumulation of citric acid requires limitation by nitrogen source in the medium (Kristiansen and Sinclair, 1978). Khare et al. (1995)
reported that the organic nitrogen sources such as peptone,
does not increase citric acid yield signicantly, while inorganic nitrogen sources, e.g. urea results 22.7% enhancement in it. To determine the eect of adding nitrogen
source on yield and productivity, two levels of 2% and
4% (w/w) were selected. Finally substrate pretreatment by
alkaline, acid and urea were conducted to compare their
impact on citric acid production. It should be mentioned
that addition of urea, NaOH and HCl has not any eect
on moisture because of drying of bagasse after treatment.
Also in urea supplementation the moisture of urea solution
was calculated to inhibit any noise in explanation of eect
of independent variables.
3. Results and discussion
The basic equation set up for statistical design was as
follows. The coecients for the eleven variables were determined by:
Ai 1=N

N
X

X i  Ki

where Ai is the coecient values, Xi is the experimental

yield, Ki is the coded value of each variable corresponding
to the respective experimental yield Xi and N is number of
experiments. Table 2 gives a comparison of the experimentally determined citric acid production yield and productivity to those predicted by solving the above equation, where
predicted yield is given by:

6990

Yi

K. Khosravi-Darani, A. Zoghi / Bioresource Technology 99 (2008) 69866993

N
X

Ai  K i

i0

for i = 0, a dummy level of +1 was used and the coecient

obtained was called A0. The standard error was determined
as the sum of the squares of the dierence between the
experimental and predicted yield for each run. The estimated error is given by:
q
S b S 2e =N
The students t-test was performed to determine the signicance of each variable employed (t-value = coecient/
Sb). Since the experiments were designed to evaluate the
relative eect of each variable on response, a signicant
level of 0.30 is acceptable (Stowe and Mayer, 1996). However, variables with a high signicant eect (at P < 0.1 and
P < 0.15) have been determined. Statistical calculations for
PBD of citric acid production from untreated sugarcane
bagasse has summarized in Table 3.
Citric acid yield (YP/S) based on the sugar consumed at
the end of fermentation were calculated. Table 3 shows statistical data for analysis of variance of citric acid production
from acid, urea and alkaline pretreated sugarcane bagasse,
respectively. Each variable which related t-value is lower
that tabulated-t (1.2 and 0.69 for P < 0 and P < 0.15) are
not signicant. Coecient values in this table show the

relative eect of each variable on citric acid productivity

and yield. The microorganism produced 94.5 g citric acid
per kg dry crude sugarcane bagasse, with yield of 97% based
on the amount of fermentable sugar consumed.
3.1. Inuence of urea pretreatment
Table 2 gives a comparison of the experimented and predicted yields and productivities of acid, urea and alkaline
pretreated sugarcane bagasse. Urea pretreatment increased
citric acid concentration of 137.6 g/kg of dry sugarcane
bagasse and citric acid yield of 96% based on sugar consumed (4th row of Table 2). Also, the maximum productivity was achieved by urea treatment to about 26.45 (g/
kg day) (5th row).
3.2. Eect of moisture content
Table 3 shows that higher values of responses (yield and
productivity) were achieved at a moisture level of 75%
(positive sign of moisture coecient in all treated and
untreated substrates). Although, lower moisture content
reduced mass transfer to the cell, and increased osmotic
pressure, but increased inter-particle space stimulate acid
production and decreased end product inhibitory in this
system.

Table 3
Statistical data for analysis of variance of citric acid yield and productivity from untreated and pretreated sugarcane bagasse
Variables

Untreated
a

Yield

A: Nitrogen source
B: Methanol (%v/w)
C: Steam time (min)
D: Sugar
(% w/w)
E: Temperature (C)
F: Time (day)
G: Solvent
H: pH
I: Seed size (cfu/ml)
J: Moisture (%)
K: Spore age (day)

Acid treated
b

Productivity

Alkaline treated
d

Yield

Productivity

Yield

Urea treated
Productivity

Yieldg

Productivityh

Coe.

t-value Coe.

t-value Coe. t-value Coe. t-value Coe.

t-value Coe. t-value Coe. t-value Coe. t-value

0.022
0.189
0.161
0.064

0.120
1.038
0.884
0.351

-0.424
2.162
3.622
2.215

-0.381
1.965
3.292
2.013

0.159
0.119
0.082
0.056

0.75
0.561
0.386
0.264

2.59
1.05
0.78
1.88

2.00
0.81
0.60
1.45

0.026
0.105
0.051
0.002

0.121
0.486
0.238
0.009

2.08
1.09
2.67
2.16

0.87
0.45
1.11
0.90

0.068
0.025
0.048
0.070

0.309
0.113
0.218
0.318

0.78
1.05
2.80
2.15

0.62
0.84
2.25
1.73

0.020
0.073
0.103
0.009
0.023
0.003
0.227

0.109
0.401
0.565
0.049
0.126
0.016
1.247

2.622
-1.060
2.944
0.497
0.869
0.264
2.332

2.383
-0.963
2.676
0.451
0.790
0.240
2.120

0.024
0.104
0.168
0.104
0.047
0.061
0.047

0.113
0.490
0.792
0.490
0.221
0.287
0.221

1.62
-0.43
5.31
0.95
1.05
2.79
-0.48

1.25
-0.33
4.11
0.73
0.81
2.16
-0.37

0.160
0.051
0.114
0.189
0.129
0.070
0.071

0.740
0.236
0.527
0.875
0.597
0.328
0.324

3.69
0.32
2.20
5.17
3.64
3.66
2.73

1.54
0.13
0.92
2.16
1.52
1.53
1.14

0.051
0.078
0.138
0.038
0.065
0.086
0.068

0.231
0.354
0.627
0.172
0.295
0.390
0.309

2.58
0.11
4.41
1.32
0.006
3.90
1.56

2.08
0.08
3.55
1.06
0.004
3.14
1.25

a
A0 0:664; (mean of the experimental yield), standard error, S b 0:182, estimated error, S 2e 0:399, tabulated t-value (degree of freedom 10) at
P < 0.1 and P < 0:15 is equal to 1.2 and 0.6, respectively.
b
A0 8:790; (mean of the experimental yield), standard error, S b 1:105, estimated error, S 2e 14:654, tabulated t-value (degree of freedom 10) at
P < 0.1 and P < 0.05 is equal to 1.3 and 1.8, respectively.
c
A0 0:774; (mean of the experimental yield), standard error, S b 0:212; estimated error, S 2e 0:541; tabulated t-value (degree of freedom 10) at
P < 0.1 and P < 0.15 is equal to 1.2 and 0.6, respectively.
d
A0 12:71 (mean of the experimental yield), standard error, S b 1:29; estimated error, S 2e 20:16, tabulated t-value (degree of freedom 10) at P < 0.1
and P < 0.05 is equal to 1.3 and 1.8, respectively.
e
A0 0:82 (mean of the experimental yield), standard error, S b 0:22, estimated error, S 2e 0:62, tabulated t-value (degree of freedom 10) at P < 0.1
and P < 0.15 is equal to 1.2 and 0.6, respectively.
f
A0 9:41 (mean of the experimental yield), standard error, S b 1:24, estimated error, S 2e 18:64, tabulated t-value (degree of freedom 10) at P < 0.1
and P < 0.05 is equal to 1.3 and 1.8, respectively.
g
A0 0:781 (mean of the experimental yield), standard error, S b 0:214, estimated error, S 2e 0:550, tabulated t-value (degree of freedom 10) at
P < 0.1 and P < 0.15 is equal to 1.2 and 0.6, respectively.
h
A0 14:84 (mean of the experimental yield), standard error, S b 2:39, estimated error, S 2e 68:81, tabulated t-value (degree of freedom 10) at P < 0.1
and P < 0.05 is equal to 1.3 and 1.8, respectively.

K. Khosravi-Darani, A. Zoghi / Bioresource Technology 99 (2008) 69866993

3.3. Eect of initial pH

As Table 3 shows (positive sign of pH coecient in all
treated and untreated substrates), the initial pH of 5.5
caused an increase in citric acid yield and productivity.
The lower value of pH was accompanied with the higher
concentration of citric acid, and the pH value increased
due to oxidation of citric acid by the fungus (Hang et al.,
1975).
3.4. Eect of methanol
Citric acid and biomass concentration, sugar consumption, citric acid yield and productivity increased in the presence of methanol. As Table 3 shows the higher amount of
methanol (4%v/w) has increased the yield and productivity
in all treated and untreated bagasse. The most famous
explanation for stimulatory inuence of methanol in citric
acid production appears is increasing of cell permeability
and decreasing end product repression (Maddox et al.,
1986). The results show that methanol concentration has
a more impact on yield than productivity.
3.5. Eect of incubation temperature and time
Temperature has a profound inuence on the fungal
production of citric acid from sugarcane bagasse. The
optimum fermentation temperature and time for citric
acid production by A. niger ATCC 9142 grown on crude
sugarcane bagasse were found to be 30 C and 5 days,
respectively. As Table 3 shows temperature does not
inuence on yield and productivity signicantly. This
result is in agree to the report of Szewezyk and Myszka
(1994) who found that the temperature did not strongly
aect the growth rate in SSF in the range of 2834 C.
Also it can be concluded from Table 3 that elongation
of incubation duration does not help to achieve a higher
value of yield and productivity. Although time increase
causes a slow increased in yield, but it may result a
decrease in productivity which is very inuential in industrial scale.
3.6. Eect of initial sugar concentration
Table 3 shows that higher initial sugar concentration
(18% w/w) results to increased yield and productivity. This
result is in agreement to Rohr who report that optimum
level for initial sugar concentration in industrial fermentations is 1422% (Rohr et al., 1983).
3.7. Eect of age of spores and inoculum size
Table 3 shows that inoculation of 6 days old spore and
inoculum size of about 105 (cfu/ml) increases yield and productivity of citric acid production from crude sugarcane
bagasse. Fernandez-Vergano et al. (1996) reported that
the sharp decrease in citric acid production observed with

6991

spores older than 7 days, and younger spores produce more

biomass and citric acid than older ones. Also increased
inoculum size, primarily, causes increased citric acid production, but further enhance of seed size is not ideal
(Fatemi and Shojaosadati, 1999).
3.8. Eect of acetone in leaching
Results of Table 3 show that, acetone has leached more
citric acid from the crude and pretreated sugarcane bagasse
than water. This result is in agree with those reported by
Hang and Woodams whose results indicate that higher
extraction eciency can be achieved by adding acetone to
leaching solvent (1989).
3.9. Eect of nitrogen supplementation
Table 3 shows that the maximum production of citric
acid was achieved by adding 4% (w/w) of urea as a source
of nitrogen to crude sugarcane bagasse. Nitrogen is usually
supplied in the form of ammonium nitrate, which was completely metabolized during fermentation periods. Citric
acid started to appear when the nitrogen concentration fell
below a low limiting value. Khare et al. also reported that
inorganic sources, e.g. urea in compared to organic sources
e.g. peptone has more eective results in increasing acid
production (1995).
3.10. Eect of sterilization time
Table 3 shows that more sterilization time (contact
of sugarcane bagasse with steam of 121 C and
15 psi) increased yield and productivity, which may be
due to increase of thermal hydrolysis of lignocellulosic
bonds and enhancing bioavailability of sugars in
substrate.
3.11. Eect of other pretreatments
Conditions for optimum citric acid production using
5 M NaOH pretreated sugarcane bagasse were initial sugar
concentration 14% (w/w); pH 5.5; moisture level 75%;
methanol concentration 4% (v/w); age of spore 6 days;
inoculum size 107 (cfu/ml); incubation time 5 days and
incubation temperature 30 C. Under these optimized conditions, 132 g citric acid was produced from 1 kg dry sugarcane bagasse with yield of 95.5% based on the amount of
fermentable sugar consumed (trial no. 9 in Table 2) and
productivity of about 25.65 g/kg day (trial no. 3). The maximal citric acid concentration, 107.68 g from 1 kg 5 N acid
pretreated sugarcane bagasse was 107.68 g. The highest citric acid yield (98.1%) and productivity (25.04 g/kg day),
were obtained at a moisture level of 75%, pH of 5.5, temperature of 30 C and initial sugar concentration of 18%
(w/w) after incubation time of 5 days, using 6 day-old
spore suspension of 107 cfu/ml in the presence of 4% (v/
w) methanol.

6992

K. Khosravi-Darani, A. Zoghi / Bioresource Technology 99 (2008) 69866993

4. Summary and conclusions

A. niger ATCC 9142 produced 94.5 g citric acid per kg
of dry crude sugarcane bagasse fermented. The maximum
yield of citric acid production was 97% based on the
amount of fermentable sugar consumed, and the maximum
productivity was about 22.22 (g/kg day).
The results showed some important aspects of citric acid
production from sugarcane bagasse by A. niger in SSF. It
can be concluded that among all pretreatments (acid, alkaline and urea) which had an important role in increasing
citric acid productivity from sugarcane bagasse, urea pretreatment was the most inuential support for acid
production.
On the basis of the results of this study, the optimum
conditions for citric acid production from urea-pretreated
sugarcane bagasse are initial sugar concentration 18% w/
w; initial pH 5.5; moisture content 75%; methanol concentration 4%v/w; sterilization time 1.5 h; inoculum size 107
(cfu/ml); age of spore 6 days; incubation time and temperature 5 days and 30 C. In these conditions process yielded
137.6 g citric acid/kg urea-pretreated dry bagasse (96%
yield based on the amount of sugar consumed) and productivity was 26.45 (g/kg day). So these conditions were
selected for doing experiment in 20 L bioreactor by ureatreated sugarcane bagasse. The produced acid concentration and yield in fermentor was 82.38 g/kg of dry substrate
and 10.47 g/kg day, respectively.
Regarding to results presented in Table 3 the most signicant variables aecting citric acid productivity from
crude sugarcane bagasse are methanol concentration,
steam time, sugar concentration, temperature, solvent
and spore age. Also the most signicant variables aecting
productivity from urea treated bagasse solvent were moisture content, temperature, steam time and sugar concentration. While the most inuential process variables for citric
acid production from alkaline and acid-treated bagasse are
solvent, moisture, sugar and acid concentration in pretreatment and moisture, incubation temperature, initial
pH and inoculum size, respectively. However, any
response to each variable depends on the selected range.
In fact in PBD or other screening designs, dummy or null
variables may occur if the dierence between two levels
of each variable is not large enough to ensure a measurable
response. Some sensitive variables on the other hand may
have their high and low levels chosen such that the size
of their dierential response is so great as to mask the eect
of other variables. Since the PBD is typically used as a preliminary optimization technique, more accurate quantitative analysis of the eect of these variables for citric acid
production is required.

Acknowledgements
We would like to thank the national nutrition and food
technology research institute (NNFTRI), and Shaheed

Beheshti Medical University (SBMU) of the I.R.Iran for

nancial support of this research project.

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