Evaluation of the Conditions Used in the Preparation of Sugarcane ...

dactylonomyskittlesΒιοτεχνολογία

12 Φεβ 2013 (πριν από 4 χρόνια και 8 μήνες)

207 εμφανίσεις

Evaluation of the Conditions Used in the Preparation of
Sugarcane Bagasse Hydrolysate for Improvement of the
Xylitol Production by Fermentative Process


Débora Danielle Virgínio da Silva
1
, Maria das Graças de Almeida Felipe
1
,
Ismael Maciel de
Mancilha
1
and Rosa Helena Luchese
2



1
Faculdade de Engenharia Química de Lorena
-

Departamento de Biotecnologia

Caixa Postal 116
-

12600
-
970 Lorena


SP
-

E
-
mail:
mgafelipe@debiq.faenquil.br

2
Universidade Fe
deral Rural do Rio de Janeiro, Departamento de Tecnologia de Alimentos

Seropédica
-

RJ


ABSTRACT



The best conditions for preparation of sugarcane bagasse hydrolysate with a view to
improve

the xylitol production by Candida guilliermondii was evaluated.

Experiments were
carried out with hydrolysate treated before or after vacuum concentration and supplemented
with rice bran autoclaved together with the hydrolysate or separately. Treating
(pH
adjustment)

the hydrolysate after its concentration and supplem
enting it with rice bran
extract solution autoclaved separately provided the maximum xylitol productivity
(Q
P
=0.51g.l
-
1
.h
-
1
) and yield (0.77g.g
-
1
). Concentrating the hydrolysate under vacuum before
treatment and autoclaving the rice bran extract solution s
eparately reduced the
volume

loss
of hydrolysate during treatment and minimised the toxic effect of the hydrolysate on the yeast,
thus improving the xylitol production.


INTRODUCTION



Large quantities of sugarcane bagasse are constantly generated by the

Brazilian agro
-
industry (Procknor, 2000). After hydrolysis, this residue presents a high amount of
fermentable carbohydrates, which could be used in bioprocesses employing microorganisms
capable of converting xylose into xylitol and other useful substance
s (Roberto
et al
., 1995;
Felipe
et al
., 1997a; Felipe
et al
., 1997b; Parajó
et al
., 1998a; Alves
et al
., 1998). Xylitol is
an anticariogenic sweetener that can be consumed by diabetics and obese people. Besides, it
prevents otitis and osteoporosis (Pepper
and Olinger, 1988; Matilla
et al
., 1998; Uhari
et al
.,
2000; Mäkinen
et al
., 2001). Because of these characteristics, xylitol is of particular interest
to food, pharmaceutical and odontological industries.


The microbiological production of xylitol by fe
rmentative process employing hydrolysates
is an alternative to the chemical synthesis, which is the conventional process for producing
xylitol commercially (Winkelhausen and Kuzmanova, 1998; Silva
et al
., 1998). Nevertheless,
the hydrolysates contain toxic

compounds, namely furfural, hydroxymethylfurfural, acetic
acid and phenolic compounds, which originate during hydrolysis (Rodrigues
et al
., 2001)
that

inhibit microbial activity (Parajó
et al
., 1998b, Palmqvist and Hahn
-
Hägerdal, 2000, Alves
et
al.
, 2002)
. Treating the hydrolysates with acids, bases and subsequently with activated
charcoal is therefore necessary
to reduce

their toxicity (Alves
et al
., 1998).

The hydrolysates
have to be concentrated under vacuum before they can be used as fermentation media
, in
order to increase the xylose concentration and consequently improve the xylitol formation
(Felipe
et al
., 1997a, Rodrigues
et al
., 2001). Supplementing the hydrolysates with nutrients
like amino acids and vitamins contained in rice bran extract improv
es the microbial growth
(Miller and Churchill, 1986).


The present study evaluated the fermentation of sugarcane bagasse hydrolysate by
Candida
guilliermondii
yeast. The hydrolysate was treated before or after the vacuum concentration
and supplemented w
ith rice bran extract prepared together with the hydrolysate or separately.


MATERIALS AND METHODS


Microorganism and Inoculum Cultivation


The experiments were conducted with
C. guilliermondii

FTI 20037 described by Barbosa
et
al

(1988). The stock cultu
re was maintained at 4°C on malt
-
extract agar slants. A medium
containing 30.0g.l
-
1

of xylose supplemented with 20.0 g.l
-
1

of rice bran extract, 2.0 g.l
-
1

of
(NH
4
)
2
SO
4

and 0.1 g.l
-
1

of CaCl
2
.2H
2
O was used for growing the inoculum. Erlenmeyer
flasks (125ml)
, each containing 50ml of medium with inoculum were incubated on a rotary
shaker (200rpm) at 30°C for 24h. Afterwards, the cells were separated by centrifugation
(2000g; 20min), rinsed twice with distilled water, and the cell cake was ressuspended in an
ad
equate volume of distilled water. The initial cell concentration in all the experiments was
0.5 g.l
-
1
.


Preparation of Hemicellulosic Hydrolysate


Sugarcane bagasse was hydrolysed in a 250l reactor at 121°C for 10min with H
2
SO
4

(solid:liquid ratio of 1:1
0). The hydrolysate was filtered, treated and vacuum concentrated.
The treatment consisted in increasing the pH to 7.0 with CaO (commercial grade), reducing it
to 5.5 with H
3
PO
4
, and adding 2.4% w/v activated charcoal (refined powder) to the
hydrolysate un
der agitation (200rpm) at 30°C for 1h. The precipitate resulting from the pH
adjustment and from the addition of activated charcoal was removed by vacuum filtration.
The vacuum concentration was performed at 70°C to triple the xylose concentration.


Medium

and Fermentation Conditions


The hydrolysate was treated either before or after the vacuum concentration, originating
two distinct hydrolysates: H1 and H2, respectively. Both hydrolysates were autoclaved at
111°C, 0.5atm and supplemented with 20.0g.l
-
1

of rice bran extract, 2.0g.l
-
1

of (NH
4
)
2
SO
4

and
0.1g.l
-
1

of CaCl
2
.2H
2
O. Next, 50 ml of each (initial pH=5.5) was placed in 125ml Erlenmeyer
flasks for fermentation at 200rpm for 54h, at 30°C.


H2, the hydrolysate that provided the maximum xylitol yield a
nd productivity, was chosen
to be used in the next experiment.


To identify the best procedure for supplementing the hydrolysate with rice bran extract,
hydrolysate treated after the vacuum concentration and supplemented with (NH
4
)
2
SO
4

and
CaCl
2
.2H
2
O wa
s supplemented either with rice bran extract solution autoclaved separately
from the hydrolysate (H2) or with rice bran extract autoclaved together with the hydrolysate
(F2). The precipitate originated during the preparation of the rice bran extract soluti
on was
removed by centrifugation at 2000g for 15min under the same conditions of temperature and
agitation described above.


Analytical Methods


Sugars (D
-
glucose, D
-
xylose, L
-
arabinose), xylitol and acetic acid were measured by high
pressure liquid chro
matography (HP1082B) using a refractive index (RI) detector, a Bio
-
Rad
HPX87H (300x7.8mm) column and 0.01N H
2
SO
4

as the eluent. The following conditions
were used: 0.6ml.min
-
1

flow rate; column temperature 45°C; detector attenuation: 16X;
sample volume 20

l. Furfural and hydroxymethylfurfural were measured using an RP
-
18
(200mm) column, CH
3
CN/H
2
O (1:8) and 1% HOAC as the eluent, 0.8ml.min
-
1

flow rate,
column temperature 25°C; ultraviolet detector. Phenolic compounds were measured
spectrometrically by the Fe
Cl
3
.6H
2
O and K
3
Fe[CN]
6

method (Kim and Yoo, 1996) at 700nm.
The cell number was directly counted in a NEUBAUER chamber (area=1/400mm
2
;
height=0.100mm).


Statistical Methods


Statistically significant differences among the conditions evaluated were determ
ined by
using a t
-
Student test.


RESULTS AND DISCUSSION



Table 1 presents the contents of sugars and toxic compounds in the sugarcane bagasse
hydrolysate
vacuum concentrated before and after treatment
, and
with

nutrient
supplementation.


Table 1
Sugarca
ne bagasse

hemicellulosic hydrolysate characteristics after treatment,
concentration and nutrient supplementation


H1
*

H2
§

F2


D
-
䝬畣潳o (g⹬
-
1
)

2.00

2.83

3.09

D
-
Xylose (g.l
-
1
)

38.00

42.38

42.18

L
-
arabinose (g.l
-
1
)

3.02

3.94

3.68

Furfural (g.l
-
1
)

0.023

0.002

0.009

Hydroxymethylfurfural (g.l
-
1
)

0.003

N.D.


丮N.



呯瑡氠l桥湯汳
n⹬
-
1
)

0.189

0.089

0.595

Acetic Acid(g.l
-
1
)

3
.50

3.52

3.49


N.D. not detected

*
H1: hydrolysate treated before concentration and supplemented with rice bran extract solution autoclaved
separately from the hydrolysate

§
H2: hydrolysate treated after concentration and supplemented with rice bran extract

solution autoclaved
separately from the hydrolysate


F2: hydrolysate treated after concentration and supplemented with rice bran extract solution autoclaved
together with the hydrolysate



As can be seen, the amounts of toxic compounds changed with changing the way the
hydrolysate was treated, concentrate
d and supplemented, except in the case of the acetic acid,
whose concentration remained almost unchanged. H2 presented the lowest rates of sugar loss
and the highest rates of furfural, hydroxymethylfurfural and total phenols removal. The
amounts of furfura
l and phenolic compounds in H2 were 4.5 and 6.7 times lower than in F2.


Alves
et al
., 1998; Silva
et al
., 1998 Rodrigues
et al
., 2001, had already demonstrated that
the lower the concentrations of toxic compounds, the higher the xylitol production. This

was
confirmed in this study. H2, the hydrolysate that presented the highest xylose consumption,
was the one with the smallest amount of toxic compounds, which shows that these substances
interfere with the xylose assimilation (Fig 1).


In all the hydrol
ysates, the yeast consumed the glucose completely in 12h of fermentation,
and part of the arabinose in a longer time (data not shown). A similar behaviour of
C.
guilliermondii

yeast in sugarcane bagasse hydrolysate was reported by Felipe
et al
., 1997a;
Fel
ipe
et al
., 1997b; Morita
et al.,
2000.
















Fig. 1
-

A
-

Xylose consumption,
B
-

Xylitol production (

) and Cell growth (
---
)
during fermentation by
C. guilliermondii

in hydrolysate treated before (H1

) or
after concentration and with rice bran
autoclaved separately (H2

) and in
hydrolysate with rice bran autoclaved together (F2

)



After 54h of fermentation, H2 (hydrolysate treated after the vacuum concentration and
supplemented with rice bran extract autoclaved separately) furnished 27.56g.
l
-
1

xylitol, which
represented an increase of around 112% in relation to the amount furnished by H1 (13.0g.l
-
1
).
H2 and F2 (hydrolysate and rice bran prepared together) gave about the same results of
xylitol production (Fig 1). H1 and H2 presented quite si
milar cell growth rates while F2
provided a cell concentration around 4.5 times higher (Fig. 1B).


Fig.2 shows that the forms of treatment, vacuum concentration and supplementation of the
hydrolysate with rice bran extract solution influenced fermentat
ive parameters.

Fig. 2
-

Xylitol Yield (Y
P/S

) and Volumetric Xylitol
Productivity (Q
P
---
) in hydrolysate treated before
concentration (H1

), hydrolysate treated after
concentration and supplemented with rice bran
autoclaved separately (H2

) and hydrolys
ate
autoclaved with rice bran extract (F2

), during
fermentation by
C. guilliermondii


0
12
24
36
48
60
0
5
10
15
20
25
30
Xylitol Formation (g.l
-1
)

Fermentation

time (h)
0
2
4
6
8
B
Cells.ml
-1
x 10
7
0
12
24
36
48
60
0
5
10
15
20
25
30
35
40
A


Xylose Consumption (g.l
-1
)
Fermentation
time (h)
12
24
36
48
60
0,0
0,1
0,2
0,3
0,4
0,5
0,6

Fermentation
time (h)
Q
P
(g.l
-1
.h)
0,0
0,2
0,4
0,6
0,8
1,0
Y
P/S
(g.g
-1
)

The xylitol production in H2 was higher than in H1 and presented meaningful differences
with probability level of the 95% (Student statistic test). The maximum resul
ts of xylitol yield
(Y
P/S
=0.77g.g
-
1
) and productivity (Q
P
=0.51g.l
-
1
.h) obtained with H2 were, respectively,
48.08% and 113% higher than those obtained with H1, probably because H1 contained more
inhibitory compounds than H2 (Table 1). The xylitol yield and

productivity values obtained
with H2 were similar to those obtained by Alves
et al
. (1998), when conducting experiments
with
C. guilliermondii

and sugarcane bagasse hydrolysate treated after vacuum concentration.


Autoclaving the rice bran extract sepa
rately from or together with the hydrolysate (H2 and
F2, respectively) had no meaningful influence (with probability level of the 95%) on the
xylitol yield. The same value (Y
P/S
=0.77g.g
-
1
) was obtained with H2 and F2 after 48h of
fermentation. So, a correl
ation of these two procedures with the fermentative parameters is
not evident. Nevertheless, H2 had an advantage over F2, since autoclaving the rice bran
extract together the hydrolysate (F2) resulted in loss of hydrolysate volume (around 35.0%),
and conse
quently in an undesirable loss of xylose.


It can be concluded that the most suitable way of preparing the sugarcane bagasse
hydrolysate for xylitol production by
C. guilliermondii
consisted in treating the hydrolysate
after vacuum concentration and supp
lementing it with rice bran extract autoclaved separately,
since with this procedure there was no loss of hydrolysate volume and the concentrations of
toxic compounds were lower.


ACKNOWLEGEMENTS



This study was financially supported by FAPESP (Fundação

de Amparo à Pesquisa do
Estado de São Paulo).
The authors are grateful to Maria Eunice Machado Coelho for revising
this paper.


REFERENCES


Alves, L.A., Michele, V., Felipe, M.G.A., Silva and J. B. A. (2002), Xylose reductase and xylitol dehydrogenase
ac
tivies of
Candida guilliermondii

as a function of different treatments of sugarcane bagasse hemicellulosic
hydrolysate employing experimental design.
Applied Biochemistry and Biotechnology,

v. 98/100, p. 403
-
413.


Alves, L.A., Felipe, M.G.A., Silva, J.B.A.
, Silva, S.S. and Prata, A.M.R.(1998), Pre treatment of sugar cane
bagasse hemicellulose hydrolysate for xylitol production by
Candida guilliermondii
.
Applied Biochemistry and
Biotechnology,
v. 70/72, p. 89
-
98.


Barbosa, M.F.S., Medeiros, M.B., Mancilha,
I.M., Schneider, H. and LEE, H. (1988), Screening of yeasts for
production of xylitol from d
-
xylose and some factors which affect xylitol yield in
Candida guilliermondii
.
Journal of Industrial Microbiology,
v
.

3, p. 241
-
251.


Felipe, M.G.A., Vitolo, M., Ma
ncilha, I.M. and Silva, S.S. (1997a), Environmental parameters affecting xylitol
production from sugar cane bagasse hemicellulosic hydrolysate by
Candida guilliermondii. Journal of Industrial
Microbiology
, v. 18, p. 251
-
254.


Felipe, M.G.A., Vitolo, M., Ma
ncilha, I.M. and Silva, S.S (1997b), Fermentation of sugar cane bagasse
hemicellulosic hydrolysate for xylitol production: effect of pH.
Biomass Bioenergy
,

v.
13, p. 11
-
14.


Kim, Y. and Yoo, Y. (1996), Peroxidase production from carrot hairy root cell cult
ure.
Enzyme Microbiology
and

Technol
ogy, v. 18, p. 531
-
535.


Makinen, K.K., Isotupa, K.P., Kivilompolo, T., Makinen, P.L., Toiveanen, J. and Soderling, E. (2001),
Comparison of erytritol and xylitol saliva stimulants in the control of dental plaque and
Mu
tans streptococci.
Caries Research
, v. 35, n. 2, p. 129
-
135.


Matilla, P.T., Knuuttila, M.L.E. and Svanberg, M.J. (1998), Dietary xylitol supplementation prevents
osteoporotic changes in streptozotocin
-
diabetic rats.
Metabolism
, v. 47, p. 578
-
583.


Miller
, T.L. and Churchill, B.W. (1986), Substrates for large
-
scale fermentation.
In: Manual of Industrial
Microbiology and Biotechnology
, Demain, A.L. and Solomon, N.A., pp. 130
-
131, American Society Microbiol.


Morita, T.A., Silva, S.S. and Felipe, M.G.A. (200
0), Effects of initial pH on biological synthesis of xylitol using
xylose
-
rich hydrolysate.
Applied Biochemistry and Biotechnology
, v. 84/86, p. 751
-
759.


Palmqvist, E. and Hahn
-
Hängerdal, B. (2000), Fermentation of lignocellulosic hydrolysates II: inhibit
ors and
mechanisms of inhibition.
Bioresource Technology
, v
.

74, p. 25
-
33.


Parajó, J.C., Domínguez, H. and Domínguez, J.M. (1998a), Biotechnological production of xylitol. Part 1.
Bioresource. Technology
, v. 65, p. 191
-
201.


Parajó, J.C., Domínguez, H. an
d Domínguez, J.M. (1998b), Biotechnological production of xylitol. Part 3.
Bioresource. Technology
, v. 66, p. 25
-
40.


Pepper, T. and Olinger, P.M. (1988), Xylitol in sugar
-

free confections.
Food Technology
, v
.

42, n. 10, p. 98
-
106.


Procknor, C. (2000),
Soluções de fábrica.
STAB


Açúcar, Álcool e Subprodutos
, v. 18, n. 4, p.14.


Roberto, I.C., Felipe, M.G.A., Mancilha, I.M., Vitolo, M., Sato, S. and Silva, S.S. (1995), Xylitol production by
Candida guilliermondii

as an approach for the utilisation of agr
oindustrial residues.
Bioresource Technology
, v
.

51, p. 255
-
257.


Rodrigues, R.C.L.B., Felipe, M.G.A., Silva, J.B.A., Vitolo, M. and Gómez, P.V. (2001), Influence of the
vacuum evaporation process associated with activated charcoal on sugarcane bagasse hem
icellulosic
hydrolysate.
Brazilian Journal of Chemical Engineering
, v. 18, n. 3, p. 299
-
311.


Silva, S.S., Felipe, M.G.A. and Mancilha, I.M. (1998), Factors that affect the biosynthesis of xylitol by xylose
-
fermenting yeasts: a review.
Applied Biochemistry

and Biotechnology
, v
.

70/72, p. 127
-
135.


Uhari, M., Tapiainen, T. and Kontiokari, T. (2000), Xylitol in preventing acute otitis media.
Vaccine
, v.8, n. 19,
p. 144
-
147.


Winkelhausen, E. and Kyzmanova, S. (1998), Microbial conversion of d
-
xylose to xylito
l.
Journal of
Fermentation and Biotechnology
, v
.

86, n. 1, p. 1
-
14.