Analysis of the Effect of Agitation and Aeration on Xylitol Production by Fermentation in Bioreactor

21 Pages·2016·0.72 MB·English

Int.J.Curr.Microbiol.App.Sci (2016) 5(6): 479-499
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 5 Number 6 (2016) pp. 479-499

Journal homepage: http://www.ijcmas.com

Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.506.055

Analysis of the Effect of Agitation and Aeration on Xylitol Production by
Fermentation in Bioreactor with Kluyveromyces marxianus Using Hydrolized
Tamarind Seed as Substrate

Ricardo Martínez-Corona1,2, Carlos Cortés Penagos2, María del Carmen Chávez-Parga3,
Mariana Alvarez-Navarrete1 and Juan Carlos González-Hernández1*

1Laboratorio de Bioquímica del Departamento de Ing. Bioquímica del Instituto
Tecnológico de Morelia, Av. Tecnológico # 1500, Colonia Lomas
de Santiaguito, C. P. 58120, Morelia, Michoacán, México
2Facultad de Químico Farmacobiología de la Universidad Michoacana
de San Nicolás de Hidalgo, México
3Posgrado de la Facultad de Ingeniería Química de la Universidad Michoacana
de San Nicolás de Hidalgo, México

*Corresponding author

A B S T R A C T

Xylitol is a compound with high industrial value due to its chemical and physical
Key words
properties; at an industrial level, xylitol is produced by catalytic hydrogenation of

Xylos e, xylose, a proc ess both costly and low-yield. As a viable alternative, xylitol can be
Ferm entation, produced by f ermentation with yeasts that use xylose as an energy source, such as
Xylit ol, Kluyveromyces marxianus. As a substrate source we proposed the use of tamarind
Aerat ion,
seed, with high xylose content in its structure. Making use of xylose by yeast plus

Agitation.
the resulting xylitol, depend on various factors among which that of greatest impact

Artic le Info is the presence of oxygen in the fermentation medium. Therefore, in this study, the

effects of aeration and agitation on xylitol production using hydrolyzed tamarind

seed as a substrate medium were ev aluated, employing a 22 factorial experimental
Accepted:
2 0 May 2016 design with a central point. In this w ay, aeration and interaction of the factors (both
A vailable Online: aeration and agitation) were the on es which had a significant effect on xylitol
1 0 June 2016 production. The highest yield reach ed was of 0.71 g of xylitol/g of xylose with

agitation at 100 rpm and, aeration at 0.1 vvm in semi anaerobic conditions.

Introduction

Xylitol is industrially produced by catalytic stages in an effort to obtain high purity
hydrogenation at both high pressure and xylose, this proved to be both expensive and
temperature, using commercial xylose or of low-yield (Gaström, 2002; Vanegas et
some similar organic source with high al., 2004). Xylitol is a carbohydrate similar
content of this monosaccharide; in said in appearance and sweetness to sucrose; it is
process, due to exhaustive purification suitable for use as a food sweetener for
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Int.J.Curr.Microbiol.App.Sci (2016) 5(6): 479-499
diabetics, as its metabolism is not regulated conditions in the medium, size of the
by insulin (Arrizon et al., 2011). Xylitol inoculum, substrate concentration and
remains stable in food preparation, neither composition of the medium, temperature and
fermenting nor producing Maillard pH; of these, the one with the most impact is
browning (Silva and Roberto, 2001); is it the presence of oxygen. At low oxygen
proved to have advantages regarding health concentration in the culture medium the
as although a sweetener it does not provoke electron transference system is incapable to
caries as it cannot be utilized or fermented reoxidaze all the NADH produced, with a
by bacteria present in the oral cavity consequent increase in the latter and a
(Bahador et al., 2012). Xylitol is an decrease in xylitol to xylulose oxidation
attractive ingredient in tablets, cough step, and finally, a considerable
mixtures, mouth-washes, tooth-paste, etc. accumulation of xylitol (Does and Bisson,
(Ahmed, 2001). 1989; Deok-Kun et al., 1997; Martinez et
al., 2002). However, there is a lack of
Xylitol can also be produced by information on the parameters that directly
fermentation of yeasts which use xylose as affect the oxygen concentration in a culture
an energy source (Ghindea et al., 2010). The medium: agitation and aeration.
first metabolic step in the fermentation of
xylose corresponds to its transport to the The biotechnological production of xylitol is
yeast cytoplasm. Xylose is introduced into of economic, academic and scientific
the cell through glucose transporters, which interest when it is obtained from low-cost
are permeases in the cell membrane that readily available raw material. Various
carry sugars by facilitated diffusion (Boris et studies have been carried out for producing
al., 2003). Alternatively, non-conventional xylitol, using residues of high xylose
yeast possesses a xylose symporter which content, to mention a few: hydrolyzed
permits its passage through the above- eucalyptus and wheat straw (Martinez et al.,
mentioned by facilitated diffusion due to the 2002), hydrolyzed rice straw (Roberto et al.,
motor force generated by a proton-ATPase 1999), hydrolyzed sugar cane (Santos et al.,
(Weusthuis and Pronk, 1994). 2003), barley malt residue (Solange and
Inés, 2005), corn cob with added xylose
Once inside the cytoplasm, the xylose is (Latina et al., 2006) and rice husk (Villalba
introduced into the yeast metabolism in et al., 2009).
three stages; first, xylose is reduced to
xylitol by D-xylose reductase (XR) using The use of a hydrolyzed medium of
NADH or NADPH as cofactor. Depending tamarind seed for xylitol production has not
on the environmental conditions, xylitol is been reported. Tamarind seed consists of a
released from the cell or oxidized to high percentage of carbohydrate (50 to 72
xylulose by xylitol dehydrogenase (XDH) in %) (El-Sidding et al., 2006), which is found
the presence of NAD+. The xylulose is then as a ramified heteropolysaccharide, known
phosphorylated by a xylulokinase with as xyloglucan, principally composed of D-
consumption of ATP, so as to be glucose, D-xylose, D-galactose and L-
incorporated in the pentose phosphate arabinose in proportions 8: 4: 2: 1,
pathway (PPP) and finally to glycolysis respectively (Kauer and Kauer, 2006). Due
(Garström, 2002). to its high xylose content, tamarind seed is a
potential substrate source with regard to
Xylitol production by fermentation depends xylitol-producing yeasts (González-
on various factors, such as: oxygen Hernández et al., 2012). As Saucedo-Luna
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et al., described, the production of alcohols and heat treatment by autoclave at 121 °C,
derived from a fermentation process from for 20 minutes. Said powder had been
organic residues takes place in three stages: previously obtained from ground and sieved
hydrolysis of the polysaccharide to form tamarind seed grain. Centrifugation at 4000
monosaccharides, fermentation of sugars by rpm for 15 minutes and filtration were used
yeast or bacteria to alcohol products and in order to remove the solid fraction of the
finally, the purification of the hydrolyzed medium.
aforementioned. Currently hydrolysis is
carried out in either concentrated acid Microorganism and preparation of
environment or in presence of enzymes. inoculum

In this study, we have evaluated the effect of The yeast employed in fermentation tests,
aeration and agitation on xylitol production Kluyveromyces marxianus ITMLB03, was
by fermentation in a bioreactor of grown in Petri plates with a medium of
hydrolyzed tamarind seed media using K. YPD-hydrolyzed tamarind seed (10g/L
marxianus, of which yeast specie very little glucose, 10g/L sugars of hydrolyzed
has been written in relation to its potential in tamarind seed, 10g/L bactopeptone, 10g/L
the production of this interesting compound. yeast extract, 10g/L agar) and stored in
refrigeration. The stored yeast was
Materials and Methods transferred by bacteriological loop to 100
mL of fermentation medium (hydrolyzed
Experimental Design tamarind seed) in a 250 mL Erlenmeyer
flask for the preparation of the inoculum,
The effect of two factors (aeration and then incubated at 30 °C for 24 h and shaken
agitation) which are key in yeast growth, at 180 rpm.
yield and production of xylitol, was
evaluated by means of a factorial 22 Fermentation Tests
experimental design with a central point.
Codified levels for each factor, the resultant Fermentation tests based on the different
four factorial treatments, plus the central treatments in the experimental design were
point are shown in Table 1. Each treatment performed in a bioreactor at a constant
of the randomized experimental design was temperature of 30 °C and an initial pH of
performed for duplicate, for statistical approximately 6.3, which was adjusted in
analysis purposes. The planning of the the tamarind seed media with a tris
experimental design and the obtaining of the NH C(CH OH) 2 M solution and was not
2 2 3
fully randomized treatments was performed controlled during tests. The initial substrate
with Stathgraphics Centurion XV® concentration (corresponding to the content
software. of reducing sugars) was adjusted by diluting
with distilled water at 50 g/L, in accordance
Hydrolyzed Tamarind Seed Media with Martínez-Corona et al., As a nitrogen
source for the yeasts, ammonium phosphate
Following the hydrolysis steps established (NH ) H PO was added, in a proportion of
4 2 2 4
by González-Hernández et al., tamarind 1 g/L.
seed powder with a maximum diameter of
0.125 mm, was transferred to acid The bioreactor employed was an
hydrolysis by diluting with nitric acid 3 % Applikon®, 3 L volume, 0.13 m diameter,
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with two Rushton 6-blades agitator, 3 Concentrations of glucose and xylose, the
deflectors and a perforated-tube air main sugars in the hydrolyzed medium and
distributor type L. This equipment was substrates for the yeast during fermentation,
sterilized for each treatment of the were calculated by enzyme tests using
experimental design, containing a medium Megazyme® kits: for glucose, the one used
culture (hydrolyzed tamarind seed) volume for catalyzed reactions of glucosidase and
of 1.7 L, autoclaved at 121 °C, 1 atm, for 15 peroxidase, and for xylose, those catalyzed
min. In this way, the hoses (for sample by xylose dehydrogenase, both with a
extraction and aeration) together with all the resultant change in absorbance of the sample
necessary material for the fermentation tests measured in a UV-Visible JENWAY 6305®
were sterilized. spectrophotometer. The measurement of
furfurals was carried out according to the
A concentration of 3 X 106 cells/mL of K. methodology proposed by Martínez et al.,
marxianus was inoculated for each The furfural, together with the
treatment. The agitator plus the pH and the hydroximethyl furfural, shows a combined
dissolved oxygen sensors were disinfected absorbance peak in the UV spectrum at 284
using ethanol and connected to the nm. The absorbance of some other
biocontroller. As previously mentioned, the compounds present in the hydrolyzed media
temperature was maintained at a constant of of organic residue is observed in the same
30 °C, by means of a water bath with a region, but maintains an absorbance code at
Science Temperature Controller®. 320 nm. By subtracting both absorbances
and comparing with a standard curve, it can
Aeration was controlled using an Aalborg be measured the concentration of these
Rotameter® connected to a compressor. The compounds in the hydrolyzed tamarind seed
flow (L/min) needed to establish the various media.
levels with regard to the aeration factor,
based on the volume of the culture medium The main fermentation products were
in the bioreactor, are shown in Table 2. quantified by using enzyme assays with
Megazyme® kits: for xylitol (regarding
Samples were taken every 12 h, which in catalyzed reactions by xylitol dehydrogenase
turn demonstrated doubling time of the yeast and diaphorase), ethanol (with alcohol
(t ), substrate consumption (glucose and dehydrogenase and aldehyde
d
xylose) and product generation (xylitol, dehydrogenase) as described by Pérez et al.,
ethanol and glycerol), resulting in the and glycerol (with enzymes glyceroquinase
conversion factor of xylose to xylitol (Y ). and lactate dehydrogenase), with a resultant
p/s
change in absorbance of the sample
Analytical Methods
measured in a UV-Visible JENWAY 6305®

spectrophotometer.
Concentration of reducing sugars from the

hydrolyzed tamarind seed media was Statistical Analysis
measured using the oxide-reduction
Analysis of the results was carried out on
technique of the DNS ( Pérez et al., 2013).
Statgraphics Centurion XV® software for
In the presence of reducing sugars, the 3, 5-
three response variables of the experimental
dinitrosalicylic acid reduces to 3-amino-5-
design: doubling time (t ), product-substrate
nitrosalicylic acid, provoking a color change d
yield (Y ) and xylitol production.
which is quantified by absorbance at a p/s
Furthermore, a Tukey-Kramer test was
wave-length of 540 nm.
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applied for the above-mentioned was these, the most important are the furfurals,
realized on said response variables together which are originated from the Maillard
with the ethanol and glycerol production, reactions regarding pentose and hexose
using JMP 6.0® software. during the acid hydrolysis; the furfural
results from the pentose dehydration while
Results and Discussion the hydroximethylfurfural from that of
xylose (Palmqvist and Hanh-Hägerdal,
The average composition of those 2000). Although the precise mechanism by
fermentation media used in distinct which the furfurals inhibit yeast metabolism
treatments of the experimental design (at is not known, it has been suggested that
bioreactor level) are shown in Table 3. The these compounds inhibit the central enzymes
initial substrate concentration for all of glycolysis (such as hexoquinase and
fermentations was of 50 g/L, according to phosphofructoquinase) or enzymes related to
the results reported previously (Martinez- either the citric acid cycle or ethanol
Corona et al., 2015). production (alcohol dehydrogenase and
aldehyde dehydrogenase) (Almeida et al.,
Hydrolyzed tamarind seed prepared in the 2007). What is more, it has been reported
conditions established by González- that yeasts not only use but also transform
Hernández et al., and adjusted to 50 g/L furfurals into furfuryl alcohol and furoic
reducing sugars (Saucedo-Luna et al., 2010) acid under anaerobic conditions
showed a higher glucose concentration (Winkelhausen et al., 2004).
(23.802 g/L) compared to that reported by
Martínez et al., for other hemicellulosic Furfural concentration in the hydrolyzed
hydrolysates: sugar cane bagasse (1.70 g/L), tamarind seed media (adjusted to 50 g/L)
rice straw (3.29 g/L), eucalyptus (1.53) and was of 0.214 g/L (Table 3), being slightly
wheat straw (2.79 g/L). As for the xylose higher than that reported by Martínez et al.,
concentration in hydrolyzed tamarind seed for hydrolyzed sugar cane bagasse (0.19
(shown in Table 3) this is seen to be slightly g/L), but lower than that obtained by the
higher than that reported by Martínez- same authors for hydrolyzed rice straw (0.27
Corona et al., for the same substrate g/L), eucalyptus wood (0.64 g/L) and wheat
prepared in similar conditions and adjusted straw (0.43 g/L). However, said
to the self-same concentration of reducing concentration of furfurals found in the
sugars, being 11.621 g/L. Despite this factor, hydrolyzed tamarind seed media did not
the xylose concentration is lower than that affect yeast growth, as a lethal inhibition
obtained by Kiyoshi et al., who also used was not observed in any of the treatments of
Candida magnoliae in fermentation of the experimental design.
hydrolyzed corncob, of approximately 25
g/L. Cell Growth

The acid and thermal conditions in which Growth and adaptation of K. marxianus in
hydrolysis is carried out, generate bioreactor fermentation tests are analyzed
compounds (such as acetic acid, phenolic based on doubling time (t ) calculated from
d
derivatives, furfural and growth kinetics in the different treatments
hydroximethylfurfural) that are capable of (data not shown). Doubling time is
both limiting and obstructing xylitol demonstrated and statistically compared in
production (Fernández et al., 2007). Of Table 4.
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According to the variance analysis regarding kinetics, reaching a product average of 5.1
the doubling time response variable, the two g/L (Figure 3A).
main factors (agitation and aeration) do not
have any significant effect on said variable. At a high agitation level (200 rpm) xylose
Factor interaction is significant on a consumption was also benefited by a high
confidence level of 95 % (Table 5). The aeration level (1 vvm), reaching 79 %
lowest doubling times were encountered at consumption, with reference to treatment 2
the highest level of both factors, as can be (aeration of 0.1 vvm) where only 58 % of
seen in Figure 1A. However, the principal available xylose was consumed (Figure 2B).
effect showed on this response variable was This fact was reflected in xylitol production
due to interaction of both factors, thereby where product concentration of 3.43 g/L was
demonstrating its significance in variance reached in treatment 1, whereas 2.58 g/L in
analysis. Therefore, the lowest doubling treatment 2 (Figure 3B). The lowest
times are found in a low agitation level (100 consumption of xylose (45 %) occurred in
rpm) combined with a high aeration one (1 treatment 4, at the central points of both
vvm), and in a high agitation level (200 factors (Figure 2B).
rpm) combined with a low aeration one (0.1
vvm), as seen in Figure 1B. The product-substrate yield (Y ) and
p/s
productivity were calculated from the xylose
The doubling times of treatments 2 and 3, consumption and xylitol production data,
resulted statistically lower according to the and are statistically compared in Table 4.
Tukey-Kramer test (Table 4). The yeast
growth in which the conditions have the Although no significant effect of the
greatest impact were at an agitation of 100 agitation and aeration on product-substrate
rpm and at an aeration of 0.1 vvm, yield (Y ) was shown in the variance
p/s
expanding the exponential growth phase analysis (Table 6), minor lineal effects of
and, therefore, increasing the doubling time both factors can be observed in Figure 4A,
response variable (4.521 h). where a better yield would be expected at
low agitation levels (100 rpm) and at low
Xylose Consumption and Xylitol aeration ones (0.1 vvm).
Production
Product-substrate yield (Table 6) was
Xylose consumption was different for the affected when a high aeration level (1 vvm)
two treatments at a low agitation level (100 was employed. At a low aeration level (0.1
rpm), at a high aeration level (1 vvm; vvm) combined with a high agitation level
treatment 5) and at a low one (0.1 vvm; (200 rpm) the lowest yield was obtained
treatment 3), as shown in Figure 2A. In (Figure 4B). As was to be expected, when a
treatment 5, little use was made of the low agitation level for aeration at 0.01 vvm
xylose available in the fermentation was used, the yield considerably increased
medium, having a final xylose uptake of and it is precisely on this point where the
only 46 %. On the other hand, in treatment 3 best results according to the experimental
accelerated substrate consumption was design, were achieved.
observed from 36 to 48 h of fermentation,
exhausting a sum-total of 70 % xylose. It The value obtained at said levels (0.7092 g
was this latter treatment which produced of xylitol/g of xylose), using the Tukey-
most xylitol at the end of fermentation Kramer test, is statistically higher than that
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of other treatments of the experimental In limited oxygen conditions, and xylose
design (Table 4). Although a high yield was being the only substrate, xylose is reduced to
not expected in treatment 1 (at 200 rpm and xylitol, but cannot be oxidized to xylose so
1 vvm) it turned out to be the second as to be incorporated to the phosphate
highest. pentose pathway (PPP), of which the end
result is the generation of pyruvate (Figure
Maximum xylitol productivity (0.106 g/L h) 5). Combined with the accumulation of
was reached in treatment 3 after 48 h of xylitol and NADH, this translates into a nil
incubation, although no statistical significant production of ATP (because of its inhibiting
difference was found in relation with the effect on the respiration chain) which is of
other treatments (Table 4). However, no vital importance in the transportation of the
relation was found between the product- monosaccharide (xylose) from the
substrate yield parameter and xylitol extracellular medium to the yeast cytoplasm
productivity: at the highest yield (in (2001). Therefore, semianaerobic conditions
treatment 5) the lowest productivity was are necessary for xylitol production, finding
observed. an improved balance in this study at an
aeration of 1 vvm.
The presence of oxygen in the culture
medium is one of the studied-factors which Although it did not have a significant effect
most affect xylitol production in a on the xylitol production response variable
fermentation process. Metabolically, in (Table 7), a slight effect of the agitation
limited oxygen conditions, an accumulation factor can be observed (Figure 5A). An
of NADH is produced (generated during the increase of the testing range of agitation
oxidation of xylitol to xylose and limiting could contribute to distinguish if a
this reaction) with subsequent xylitol significant effect of this factor does, in fact,
accumulation (Deok-Kun et al., 1997). exist. In comparing interaction effects,
higher xylitol production can be observed
It was precisely the aeration factor which with low-level agitation again (Figure 5B).
had a significant effect on the xylitol
production at the end of the fermentation, as In spite of the fact that treatment 3 had the
shown in the variance analysis for this highest production of xylitol, this did not
response variable in Table 7. Nevertheless, differ, statistically speaking, from the other
the effect of this factor was not that treatments of the experimental design
expected, but quite the opposite. Xylitol (compared using the Tukey-Kramer test in
production was seen to be affected at a low Table 4), owing to the variability of the
level of 0.1 vvm, whereas it was benefited results obtained from the repetition of the
from 1 vvm (Figure 5A). fermentation tests.

It has been reported that xylose metabolism,
As has been already mentioned, the highest
in yeasts such as K. marxianus, would not
yield reached in treatments at bioreactor
appear to be limited by its transportation
level with K. marxianus, were at low
from extracellular medium to cytoplasm.
agitation (100 rpm) and aeration levels (0.1
However, the transportation of xylose
vvm), being 0.71 g of xylitol/g of xylose.
through the cellular membrane could limit
This value is much more than that obtained
its use in aerobic and semianaerobic
by Wannawilai et al., (2007) who employed
conditions (Winkelhausen et al., 2004).
C. magnoliae in a bioreactor (fermentation

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volume of 1 L), in a synthetic fermentation With agitation at 100 rpm, ethanol
medium of xylose, which resulted in 0.39 g production increased during the initial hours
of xylitol/g of xylose; furthermore, it is of fermentation at both levels of aeration
higher than that obtain by Furlan and de (Figure 7A). Of these two treatments, the
Castro (2001), using C. parapsilosis in one at aeration of 0.1 vvm was that which
fermentation at bioreactor level with mixed showed higher product generation, which
synthetic medium which was 0.63 g of was maintained at a constant as from
xylitol/g of xylose. approximately 24 h. At a high aeration level
(treatment 3), there were slight variations as
One of the highest yields obtained at from 12 h, reaching maximum concentration
bioreactor level for xylitol production, was at the end of fermentation.
that attributed to Ojamo et al., (1994) which
was of 0.78 g of xylitol/g of xylose by At a high agitation level, treatments 1 and 2
fermentation in a synthetic medium of showed a similar ethanol production after 80
xylose, using C. guilliermondii. There have h of fermentation. On the other hand, at a
also been reports of high yields based on low agitation level, the highest production
fermentation from different sources of occurred during the initial hours (Figure
hemicellulosic hydrolysates using C. 7B). With aeration at 1 vvm, ethanol
guilliermondii at bioreactor level: Roberto et concentration maintained constant during
al., (1999) reached a yield of 0.73 g of the remainder of the kinetic; while a low
xylitol/g of xylose, employing rice straw as aeration level (0.1 vvm) showed a maximum
hydrolyzed substrate medium; while for at 36 h. Contrasting with the afore-
their part, Acosta et al., (2005) reported a mentioned treatments, in the central point
yield of 0.75 g of xylitol/g of xylose by the greatest production of ethanol occurred
fermentation of hydrolyzed sugar cane at 24 h of fermentation (Figure 7B).
bagasse. In our study, the yield reached does
not differ all that much from the above- Tendencies observed in Figure 7, where the
mentioned reports with a widely-used yeast highest production of ethanol in all the
employed for xylitol production, namely C. treatments of the experimental design
guilliermondii, at bioreactor level, proposing occurred in the first hours of fermentation,
K. marxianus as a yeast with acceptable would suggest that ethanol production is
yields in xylitol production. principally linked to the exponential growth
phase of the yeast (data not shown) and,
Glucose Consumption and Ethanol consequently, to glucose consumption in the
Production fermentation medium. However, xylose
uptake and metabolism could improve
No significant differences were observed ethanol production in any growth phase.
with respect to glucose consumption in the 5
treatments of the experimental design Xylose consumption together with xylitol
(Figure 6). In all cases, consumption of this production during the stationary phase, limit
substrate was accelerated during first 24 h of ethanol production, for which reason the
fermentation, followed by a period of concentration of said product is maintained
deceleration (under 5 g/L). Total yeast at a constant during the mentioned growth
consumption of available glucose in the stage of K. marxianus.
different treatments was between 92 and 94
%.

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Table.1 Treatments and coded values for the factorial 22 experimental
design with a central point.

Coded values
Agitation Aeration
Treatment
(rpm) (vvm)
A B
1 200 1 1 1
2 100 1 -1 1
3 200 0.1 1 -1
4 150 0.55 0 0
5 100 0.1 -1 -1

Table.2 Air flow in the rotameter for each level of the factorial 22 experimental
design with a central point.

Aeration (vvm) Coded value Air flow (L/min)
0.1 -1 0.17
0.55 0 0.935
1 1 1.7

Table.3 Composition of the hydrolyzed tamarind seed media for the treatments
of the factorial 22 experimental design with a central point.

Initial substrate
Glucose (g/L) Xylose (g/L) Furfurals (g/L)
concentration (g/L)
50 23.802 ± 3.398 12.795 ± 0.905 0.214 ± 0.014
The data show the mean ± standard deviation for n=10

Table.4 Average results of factorial 22 experimental design with a central point, for different
parameters compared by Tukey-Kramer statistical test.

Y (g Xylitol
Treat Factor Factor p/s Xylitol Ethanol Glycerol
xylitol/g t (h) productivity
ment A B d (g/L) (g/L) (g/L)
xylose) (g/L h)
1 1 1 0.5609B 3.648AB 3.675A 0.077A 13.442B 1.627B
2 -1 1 0.4491C 2.563BC 2.579A 0.054A 12.366B 3.79A
3 1 -1 0.4805BC 2.337C 5.101A 0.106A 15.270B 1.834B
4 0 0 0.3931C 3.702AB 2.566A 0.053A 14.152B 2.011B
5 -1 -1 0.7092A 4.521A 2.378A 0.050A 21.538A 2.78AB
Means followed by the same letter do not differ at a significance level α=0.05. Table representative for n=2

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Table.5 ANOVA for the doubling time (td) response variable

Degrees of
Source of Sum of Mean Square
Freedom F-value P-value
variation Squares (SS) (MS)
(df)
A:Agitation 0.20924 1 0.20924 1.64 0.2481
B:Aeration 0.603022 1 0.603022 4.72 0.0729
AB 5.34383 1 5.34383 41.79 0.0006
Error 0.767207 6 0.127868

Total 6.9233 9

The null hypothesis are rejected when the P-value is less than the established significance level (α=0.05)

Table.6 ANOVA for the product-substrate yield (Yp/s) response variable

Mean
Source of Sum of Degrees of
Square F-value P-value
variation Squares (SS) Freedom (df)
(MS)
A:Agitation 0.0161666 1 0.0161666 1.91 0.2161
B:Aeration 0.00683732 1 0.00683732 0.81 0.4032
AB 0.0579722 1 0.0579722 6.85 0.0397
Error 0.0507482 6 0.00845804

Total 0.131724 9

The null hypothesis are rejected when the P-value is less than the established significance level (α=0.05)

Table.7 ANOVA for the xylitol production response variable

Degrees of
Source of Sum of Mean Square
Freedom F-value P-value
variation Squares (SS) (MS)
(df)
A:Agitation 0.750068 1 0.750068 0.97 0.3637
B:Aeration 7.28894 1 7.28894 9.38 0.0221
AB 1.32243 1 1.32243 1.7 0.2398
Error 4.66054 6 0.776757

Total 14.022 9

The null hypothesis are rejected when the P-value is less than the established significance level (α=0.05)
488