Optimization of inulinase production by solid-state

fermentation using sugarcane

bagasse as substrate
Marcio Mazutti, Joao Paulo Bender, Helen Treichel, Marco Di Luccio
Universidade Regional Integrada do Alto Uruguai e das Missoes Campus de Erechim,
Departamento de Engenharia de Alimentos, Av. Sete de Setembro 1621,
Erechim 99700-000, Brazil

Abstract
The production of enzymes by bioprocesses is a good alternative to add value to agroindustry residues. Sugarcane bagasse is an abundant
by-product of sugar industry and was tested as support and carbon source for production of inulinase (2,1--d-fructanohydrolase, E.C. 3.2.1.7)
from Kluyveromyces marxianus NRRL Y-7571 by solid-state fermentation. Corn steep liquor was used as nitrogen supplement. Factorial design
and response surface analysis were carried out to evaluate the effects of temperature (30.441.6 C) and corn steep liquor (1327.1%, w/v) on the
production of inulinase. Optimum fermentation conditions were found to be: 36 C and 20 wt.% of corn steep liquor. Under optimized conditions,
the extra-cellular enzyme concentration reached 391.9 U/g of dry fermented bagasse.
Keywords: Inulinase; Optimization; Solid-state fermentation; Sugarcane; Bagasse

1. Introduction
Solid-state fermentation (SSF) may be defined as a fermentation process where the microorganisms grow in solid substrates
with low water concentration [13]. Many studies about the
application of SSF are focused in adding value to agroindustry
residues, which have been extensively used as physical support
or source of nutrients in SSF [2,47].
Brazil is known as one of the greatest producers of sugar
from sugarcane in the world [8]. Sugarcane production in 2004
was 410 million t per month. The crop is mainly directed for
production of ethyl alcohol, sugar and spirits [9]. Sugarcane
bagasse is a by-product resulting from juice extraction. This
waste basically consists of 50% of cellulose, 30% sugars and
2.4% of ashes [7].
The production of enzymes by SSF has gained much attention
in biotechnology studies for production of lipases [5], inulinases
[2], proteases [10], etc. The use of low cost residues, higher
productivities, low energy requirements, lower wastewater pro-

Corresponding author. Tel.: +55 54 520 9000; fax: +55 54 520 9090.
E-mail address: diluccio@uricer.edu.br (M.D. Luccio).

duction, extended stability of products and low production costs

are some of the main advantages of SSF [3,11]. The selection
of a suitable microorganism is an important aspect of SSF for
production of enzymes [3]. The microorganism should be able
to grow at low water activity, to be GRAS (Generally Recognized as Safe) and be accepted by FDA (Food and Drug
Administration) [12].
Inulinase production by Kluyveromyces marxianus NRRL Y7571 is then of great interest, since it attends the requirements
of GRAS and just a few studies of the production of inulinase
by SSF using this yeast are reported [2].
Inulinases are potentially useful enzymes for production of
high fructose syrups (HFS) from inulin [13]. Fructose production by inulin hydrolysis is more advantageous than conventional
process based on starch, which includes the action of -amylase,
amyloglucosidase and glucose isomerase, yielding only 45% of
fructose in the final product due to the thermodynamical equilibrium of the reaction. Inulinase based hydrolysis of inulin can
yield products with 95% of fructose [14].
This work aimed to optimize inulinase production by SSF
using sugarcane bagasse and corn steep liquor as substrates.
The optimization was carried out by experimental design and
surface analysis methodology.

2. Materials and methods

2.1. Microorganism and media
The strain of K. marxianus NRRL Y-7571 was maintained at 4 C on YM
agar medium containing (g L1 ): yeast extract 3.0, malt extract 3.0, peptone 5.0,
glucose 10.0, agar 20.0 and subcultured every 3 weeks.
Cell production for pre-inoculum was carried out in 50 mL test tubes with
10 mL of liquid YM medium. The medium was inoculated with a loopful of
stock culture and incubated at 30 C for 24 h. This suspension was used for
inoculation without any further treatment.
Medium for inoculum contained (g L1 ): sucrose 20.0, yeast extract 5.0,
K2 HPO4 5.0, NH4 Cl 1.5, KCl 1.15 and MgSO4 7H2 O 0.65. Each test tube with
YM medium was transferred to a 500 mL Erlenmeyer flask with 100 mL of
medium and incubated at 30 C and 150 rpm for 24 h.

50 C. After reaction, the reducing sugar concentration is determined by DNS

method [15]. One unit of inulinase activity is defined as the amount of enzyme
necessary to hydrolyze 1 mol of sucrose per minute at reaction conditions.
Inulinase has different hydrolytic activity on sucrose when compared to inulin.
This behavior may be represented by the ratio of activity on sucrose/activity on
inulin (S/I). Value of S/I lower than 50 is characteristic of inulinase behavior
[16]. The optimization was carried out determining only the hydrolytic activity
on sucrose. The activity on inulin was determined only in the optimum condition
(central point).

2.6. Microbial growth

As an indirect way to quantify microbial growth, glucosamine was assayed
in the solid according to Aidoo et al. [17].

3. Results and discussion

2.2. Solid-state fermentation

Sugarcane bagasse from a local industry (Cooperativa Tritcola Erechim
Ltd.) was used as substrate for inulinase production. Fermentations were carried out in conical flasks (500 mL) with 5 g of dry bagasse, supplemented with
corn steep liquor (CSL, Corn Products, Brazil) in concentrations defined by the
experimental design. Moisture was adjusted to 65%, each flask was covered
with hydrophobic cotton and autoclaved at 121 C for 20 min. Preliminary studies showed that no changes in moisture content of the substrate after autoclaving
were detected. After cooling, each flask was inoculated with 3 mL of the suspension previously prepared and incubated for 72 h in a chamber with temperature
and humidity control.
A 22 full factorial design was performed to assess the effect of supplement
concentration and incubation temperature on inulinase production. A central
point was carried out in triplicate plus two axial points (star configuration,
= 21/2 ) for each independent factor for experimental error evaluation and
second-order effects estimation, respectively. Table 1 shows the range of the
studied factors and the correspondent coded levels.

2.3. Statistical analysis

Statistica (version 5.1) software was used for regression and graphical analyses of the data obtained. The statistical significance of the regression coefficients
was 95%. The optimum concentrations of the variables were obtained by the
graphical analysis.

2.4. Extraction of the enzyme

After fermentation the whole sample of each flask was extracted by the addition of 50 mL of sodium acetate buffer 0.1 mol L1 pH 4.8, following incubation
at 30 C and 150 rpm for 30 min. Enzyme activity was assayed in the supernatant
after vacuum filtration (Whatman qualitative filter paper, grade 1) and the solid
matter was assayed for glucosamine and total reducing sugar (TRS) by DNS
method [15]. Glucosamine, inulinase activity and TRS were expressed based
on the mass of dry substrate. All analyses were carried out in triplicates and
standard deviation was lower than 5%.

Table 2 presents the matrix of the complete factorial design

with the correspondent responses in terms of enzyme activity. The results show that the highest enzyme production
(391.9 U g1 ) was obtained at central point conditions (36 C
and 20 wt.% of CSL). This production is higher than the reported
in literature for production of inulinase by solid-state fermentation using wheat bran (122.9 U g1 ) [2]. Production of inulinase
as high as 176 U mL1 in submerged fermentations have been
reported [18].
The optimum incubation temperature for inulinase production found in our work was 36 C, similar to that suggested
by Selvakumar et al. (37 C), when cultivating K. marxianus
CDBB-L-278 in solid-state fermentations [2]. Other studies also
found similar results in submerged fermentations with yeast of
the genus Kluyveromyces [15].
Statistical analysis of the results in Table 2 was performed
yielding an empirical coded model of inulinase activity in function of temperature and corn steep liquor concentration. Linear
and quadratic parameters for the concentration of CSL were not
statistically significant (p < 0.05) and were added to the lack of
fit.
The optimized coded model for inulinase activity was validated by analysis of variance (ANOVA), presented in Table 3.
The ANOVA shows a high R (0.86, correlation coefficient) and a
good performance of the F-test for regression (calculated values
Table 2
Matrix of the experimental design (coded and values) with responses in terms
of inulinase activity (after 72 h of fermentation)
Run

Corn steep liquor

(% w/w)

Temperature ( C)

Activity (U gds1 )

1
2
3
4
5
6
7
8
9
10
11

1 (15%)
+1 (25%)
1 (15%)
+1 (25%)
1.41(13%)
+1.41(27.1%)
0 (20%)
0 (20%)
0 (20%)
0 (20%)
0 (20%)

1 (32 C)
1 (32 C)
+1 (40 C)
+1 (40 C)
0 (36 C)
0 (36 C)
1.41(30.4 C)
+1.41(41.6 C)
0 (36 C)
0 (36 C)
0 (36 C)

238.97
246.13
194.41
220.87
239.47
224.46
19.01
26.08
327.54
315.72
391.94

2.5. Enzyme activity assay

Enzyme activity was determined by initial reaction rate of reducing sugar
production under controlled conditions. The method consists in adding 0.5 mL
of enzyme extract to 4.5 mL of sucrose solution (2% w/w) in 0.1 mol L1 pH
4.8 sodium acetate buffer and incubating the reaction medium for 10 min at
Table 1
Range of the factors investigated in the experimental design
Level
Temperature ( C)
Corn steep liquor (% w/w)

1.41
30.4
13

1
32
15

0
36
20

+1
40
25

+1.41
41.6
27.1

gds = grams of dry substrate.

Table 3
ANOVA for inulinase activity as response
Source of
variation

Sum of
squares

Degrees of
freedom

Regression
Residual
Lack of fit
Pure error

100046.1
30288.6
26923.1
3365.5

1
9
7
2

Total

130334.7

10

Mean square

F-test

100046.1
3365.4

29.7

Regression coefficient: R = 0.86; F0.95;1;9 = 5.12.

about 5.8 times the listed one). Therefore, Eq. (1) is predictive
of inulinase production in the investigated range of factors, and
consists in a second-order function for temperature. This model
is represented in Fig. 1:
activity = 314.9 127.8T 2

(1)

The response surface in Fig. 1 shows that the maximum inulinase activity is obtained in the region of central point. It is also
clear that the concentration of CSL does not influence inulinase
production in the investigated range. When incubation temperature was set at the extremes of the studied interval low values
of inulinase activity were obtained, since low temperature may
lead to reduction in metabolism of the microorganism and high
temperature may induce enzyme inactivation.
After optimization, fermentation kinetics was determined at
the optimized conditions, as observed in Fig. 2. The results show
a decrease of 40% in total reducing sugars (TRS) in the first
6 h of fermentation, following stabilization. The production of
enzyme is not detected before 12 h of fermentation and total consumption of sugars during fermentation was not observed. This
latter fact may be related to the occurrence of partial hydrolysis of cellulose during the step of hydrolysis of sucrose in the
determination of total reducing sugar. Cellulose is a polymer of
glucose bound by (1 4) glycosidic bonds, which might not
be readily accessible to the yeast but still quantified by the analytical method [19]. Thus, the residual TRS observed in Fig. 2
may be resultant of this partial hydrolysis of cellulose during

Fig. 2. Kinetics of inulinase production at optimum conditions (36 C, 20% w/w

of corn steep liquor). TRS = total reducing sugars, gds = grams of dry substrate.

TRS quantification, and it was not available to the yeast during

fermentation.
Maximum inulinase activity occurred after 96 h of fermentation. Selvakumar et al. [2] report that the maximum production of the enzyme by SSF occurs after 72 h for K. marxianus
CDBB-L-278 (122.8 U g1 ) and after 48 h for Staphylococcus
sp. (107.6 U g1 ) [2].
Fig. 2 also shows the kinetics of glucosamine production as
an indirect means to quantify the growth of microorganism. An
increase in glucosamine content is observed during fermentation, what is a possible indication of cell growth. Glucosamine
results also suggest that production of inulinase is associated
to growth, which agrees with the results for production of
inulin by submerged fermentation reported by other studies
[20].
Inulinase productivity obtained in our work was 3.34 U g1
h1 , almost two times the highest productivity reported in literature, 1.71 U g1 h1 [2].
4. Conclusions
In this study optimization of inulinase production by K. marxianus NRRL Y-7571 using sugarcane bagasse as substrate was
carried out. The best fermentation conditions found after optimization was 36 C and 20% of corn steep liquor, which yielded
about 390 U g1 . Maximum productivity was 3.34 U g1 h1 ,
the highest reported in literature to date.
Sugarcane bagasse seems to present a great nutritional potential for growth of K. marxianus NRRL Y-7571 and production
of inulinase.
Acknowledgements
Authors are grateful to Laboratorio de Engenharia de Bioprocessos/UNICAMP for supplying the microorganism used in
this work.
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Fig. 1. Response surface for inulinase activity.

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