Optimization of inulinase production by solid-state

fermentation using sugarcane

bagasse as substrate
Marcio Mazutti, João Paulo Bender, Helen Treichel, Marco Di Luccio ∗
Universidade Regional Integrada do Alto Uruguai e das Missões 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 (30.4–41.6 ◦ C) and corn steep liquor (13–27.1%, 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 duction, extended stability of products and low production costs

Solid-state fermentation (SSF) may be defined as a fermenta-
tion process where the microorganisms grow in solid substrates
with low water concentration [1–3]. 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 [2,4–7].
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).
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 Rec-
ognized as Safe”) and be accepted by FDA (“Food and Drug
Administration”) [12].
Inulinase production by Kluyveromyces marxianus NRRL Y-
7571 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 produc-
tion 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 equi-
librium 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 L−1 ): 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 L−1 ): 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.
2.2. Solid-state fermentation
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

Sugarcane bagasse from a local industry (Cooperativa Tritı́cola Erechim

Table 2 presents the matrix of the complete factorial design
Ltd.) was used as substrate for inulinase production. Fermentations were car- with the correspondent responses in terms of enzyme activ-
ried out in conical flasks (500 mL) with 5 g of dry bagasse, supplemented with ity. The results show that the highest enzyme production
corn steep liquor (CSL, Corn Products, Brazil) in concentrations defined by the (391.9 U g−1 ) was obtained at central point conditions (36 ◦ C
experimental design. Moisture was adjusted to 65%, each flask was covered and 20 wt.% of CSL). This production is higher than the reported
with hydrophobic cotton and autoclaved at 121 ◦ C for 20 min. Preliminary stud-
ies showed that no changes in moisture content of the substrate after autoclaving
in literature for production of inulinase by solid-state fermenta-
were detected. After cooling, each flask was inoculated with 3 mL of the suspen- tion using wheat bran (122.9 U g−1 ) [2]. Production of inulinase
sion previously prepared and incubated for 72 h in a chamber with temperature as high as 176 U mL−1 in submerged fermentations have been
and humidity control. reported [18].
A 22 full factorial design was performed to assess the effect of supplement The optimum incubation temperature for inulinase produc-
concentration and incubation temperature on inulinase production. A central
point was carried out in triplicate plus two axial points (star configuration,
tion found in our work was 36 ◦ C, similar to that suggested
α = ± 21/2 ) for each independent factor for experimental error evaluation and by Selvakumar et al. (37 ◦ C), when cultivating K. marxianus
second-order effects estimation, respectively. Table 1 shows the range of the CDBB-L-278 in solid-state fermentations [2]. Other studies also
studied factors and the correspondent coded levels. found similar results in submerged fermentations with yeast of
the genus Kluyveromyces [15].
2.3. Statistical analysis Statistical analysis of the results in Table 2 was performed
yielding an empirical coded model of inulinase activity in func-
Statistica (version 5.1) software was used for regression and graphical analy-
ses of the data obtained. The statistical significance of the regression coefficients tion of temperature and corn steep liquor concentration. Linear
was 95%. The optimum concentrations of the variables were obtained by the and quadratic parameters for the concentration of CSL were not
graphical analysis. statistically significant (p < 0.05) and were added to the lack of
fit.
2.4. Extraction of the enzyme The optimized coded model for inulinase activity was vali-
dated by analysis of variance (ANOVA), presented in Table 3.
After fermentation the whole sample of each flask was extracted by the addi-
tion of 50 mL of sodium acetate buffer 0.1 mol L−1 pH 4.8, following incubation
The ANOVA shows a high R (0.86, correlation coefficient) and a
at 30 ◦ C and 150 rpm for 30 min. Enzyme activity was assayed in the supernatant good performance of the F-test for regression (calculated values
after vacuum filtration (Whatman qualitative filter paper, grade 1) and the solid
matter was assayed for glucosamine and total reducing sugar (TRS) by DNS Table 2
method [15]. Glucosamine, inulinase activity and TRS were expressed based Matrix of the experimental design (coded and values) with responses in terms
on the mass of dry substrate. All analyses were carried out in triplicates and of inulinase activity (after 72 h of fermentation)
standard deviation was lower than 5%.
Run Corn steep liquor Temperature (◦ C) Activity (U gds−1 )
(% w/w)
2.5. Enzyme activity assay
1 −1 (15%) −1 (32 ◦ C) 238.97
Enzyme activity was determined by initial reaction rate of reducing sugar 2 +1 (25%) −1 (32 ◦ C) 246.13
production under controlled conditions. The method consists in adding 0.5 mL 3 −1 (15%) +1 (40 ◦ C) 194.41
of enzyme extract to 4.5 mL of sucrose solution (2% w/w) in 0.1 mol L−1 pH 4 +1 (25%) +1 (40 ◦ C) 220.87
4.8 sodium acetate buffer and incubating the reaction medium for 10 min at 5 −1.41(13%) 0 (36 ◦ C) 239.47
6 +1.41(27.1%) 0 (36 ◦ C) 224.46
Table 1 7 0 (20%) −1.41(30.4 ◦ C) 19.01
Range of the factors investigated in the experimental design 8 0 (20%) +1.41(41.6 ◦ C) 26.08
9 0 (20%) 0 (36 ◦ C) 327.54
Level −1.41 −1 0 +1 +1.41 10 0 (20%) 0 (36 ◦ C) 315.72
Temperature (◦ C) 30.4 32 36 40 41.6 11 0 (20%) 0 (36 ◦ C) 391.94
Corn steep liquor (% w/w) 13 15 20 25 27.1
gds = grams of dry substrate.
Table 3
ANOVA for inulinase activity as response
Source of Sum of Degrees of Mean square
variation squares freedom
Regression 100046.1 1 100046.1
Residual 30288.6 9 3365.4
Lack of fit 26923.1 7
Pure error 3365.5 2
Total 130334.7 10
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
The response surface in Fig. 1 shows that the maximum inuli-
nase 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 temper-
ature 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 con-
sumption of sugars during fermentation was not observed. This
latter fact may be related to the occurrence of partial hydroly-
sis 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 ana-
lytical method [19]. Thus, the residual TRS observed in Fig. 2
may be resultant of this partial hydrolysis of cellulose during
Fig. 1. Response surface for inulinase activity.
F-test
29.7
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
(1) fermentation.
Maximum inulinase activity occurred after 96 h of fermen-
tation. Selvakumar et al. [2] report that the maximum produc-
tion of the enzyme by SSF occurs after 72 h for K. marxianus
CDBB-L-278 (122.8 U g−1 ) and after 48 h for Staphylococcus
sp. (107.6 U g−1 ) [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 fermenta-
tion, 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 g−1
h−1 , almost two times the highest productivity reported in liter-
ature, 1.71 U g−1 h−1 [2].
4. Conclusions
In this study optimization of inulinase production by K. marx-
ianus NRRL Y-7571 using sugarcane bagasse as substrate was
carried out. The best fermentation conditions found after opti-
mization was 36 ◦ C and 20% of corn steep liquor, which yielded
about 390 U g−1 . Maximum productivity was 3.34 U g−1 h−1 ,
the highest reported in literature to date.
Sugarcane bagasse seems to present a great nutritional poten-
tial for growth of K. marxianus NRRL Y-7571 and production
of inulinase.
Acknowledgements
Authors are grateful to Laboratório de Engenharia de Bio-
processos/UNICAMP for supplying the microorganism used in
this work.
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