Natural Science, 2009, 1, 47-54 NS
http://dx.doi.org/10.4236/ns.2009.11009
Adaptation fermentation of Pichia stipitis and combination
detoxification on steam exploded lignocellulosic
prehydrolyzate
Jun-Jun Zhu1, Qiang Yong1, Yong Xu1, Shang-Xing Chen2, Shi-Yuan Yu1*
1Ministry of Education, Key Laboratory of Forest Genetics & Biotechnology, Nanjing Forestry University, Nanjing 210037, China;
2College of Forestry, Jiangxi Agricultural University, Nanchang 330045, China. *Corresponding author: Tel: [phone]; Fax:
[phone].
Received 25 May 2009; revised 5 June 2009; accepted 8 June 2009.
ABSTRACT
Yeast Pichia stipitis CBS 5776 was developed
through adaptation fermentation step by step in
steam exploded corn stover prehydrolyzate
because high concentration of weak acids and
other inhibitors present in the prehydrolyzate
could degrade the fermentability. However, the
adaptability of Pichia stipitis CBS 5776 in the
prehydrolyzate was so limited that steam strip-
ping and overliming were applied to remove
these inhibitors from it. Corn stover was steam
exploded; the filtrate of steam exploded corn
stover was hydrolyzed with dilute sulfuric acid,
and then the acid hydrolyzate was detoxified
and fermented by Pichia stipitis CBS 5776.
Steam stripping could remove volatile com-
pounds from the acid hydrolyzate and the fil-
trate. At a steam stripping time of 120min, 81%
acetic acid and 59% formic acid were removed
from the acid hydrolyzate, 77% acetic acid and
45% formic acid were removed from the filtrate,
while furfural was stripped off completely from
the acid hydrolyzate and the filtrate. Overliming
could reduce the contents of furfural and phe-
nolics present in the acid hydrolyzate, however,
sugars, especially pentoses, were also removed
partially. It was necessary to detoxify the acid
hydrolyzate in order to ferment the sugars to
ethanol. Acid hydrolyzate detoxified with a
combination of steam stripping for 120 min and
overliming at pH11 and 60℃ for 90 min, its fer-
mentability was significantly improved. Xylose
was consumed nearly completely in 24h with an
ethanol yield of 15.92g/l, 80.34% of theoretical.
Keywords: Steam Explosion; Steam Stripping;
Overliming; Inhibitor; Acid Hydrolyzate
1. INTRODUCTION
For several decades, ethanol has been promoted as a
promising alternative fuel for transportation. The use of
fossil fuels has contributed to the buildup of carbon di-
oxide in the atmosphere; however ethanol is a clean-
burning fuel that makes no net contribution to global
warming because the carbon dioxide produced by the
combustion of ethanol is consumed by plant growth
which continues the carbon cycle balance in the nature.
Ethanol can be produced from any material containing
simple or complex sugars, such as sugar cane and vari-
ous starchy materials (corn, wheat, and potatoes). How-
ever, the most promising raw material is represented by
lignocellulose because of its renewable nature, abun-
dance and low cost [1].
Lignocelluloses are mainly comprised of cellulose,
hemicellulose, and lignin. The substrate must be pre-
treated so that it is more amenable to conversion. A
number of pretreatments such as dilute acid hydrolysis
[2], alkali treatment [3], sodium sulphite treatment [4],
steam explosion [5], ammonia fiber explosion [6], lime
treatment [7], wet oxidation [8], liquid hot water pre-
treatment [9], organic solvent treatment [10], and biolo-
gial pretreatment [11], and so on, have been used fre-
quently to improve the saccharification of the carbohy-
drates. Of these methods, steam explosion has been rec-
ognized as one of the most cost- effective and potential
pretreatment methods [12,13]. During the pretreatment
process with high temperature, a part of cellulose, hemi-
cellulose and lignin is degraded to produce compounds
which inhibit enzymatic hydrolysis and ethanol fermen-
tation. The main degraded products from steam exploded
corn stover have been identified by HPLC and GC-MS
analysis [14].
The inhibiting compounds are divided into three main
groups based on origin: weak acids, furan derivatives,
and phenolic compounds. Weak acids (formic, acetic,
and levulinic acid), furan derivatives (furfural, 5-hy-

48 J. J. Zhu et al. / Natural Science 1 (2009) 47-54
droxymethylfurfural) from sugar degradation, phenol
compounds from lignin degradation are considered to be
potential fermentation inhibitors from pretreated ligno-
cellulose [15]. In order to improve the fermentability of
yeast, it can be done from the following two aspects, one
is to make the yeast to adapt to the prehydrolyzate step
by step, and another is to adopt methods to remove the
inhibitors from the prehydrolyzate. Nigam investigated a
mutant Pichia stipitis NRRL Y-7124 to adapt in acid
hydrolyzate. When it was tested for its ability to ferment
acid hydrolyzate, it showed shorter fermentation time,
better tolerance to acid and could ferment at lower pH.
The ethanol yield and productivity were increased 1.3
and 2.1 fold, respectively [16]. However, more research-
ers investigated detoxification methods to detoxify the
hydrolyzate to improve fermentation efficiency. Detoxi-
fication methods include neutralization [17], overliming
[18], steam stripping [19], ion-exchange [20], treatment
with activated carbon [20], wood charcoal [21], and lac-
case and peroxidase [22]. In many instances, the most
economical and widely used method of detoxification
involves treatment of hydrolyzates with solid calcium
hydroxide [23]. This process of “overliming” is reported
as an effective method of reducing toxicity of various
hydrolyzates [24]. However, different yeasts endure the
ability of inhibitors differently. Previous investigation in
this laboratory indicated that only “overliming” was not an
effective method to detoxify hydrolyzate fermented by
Yeast Pichia stipitis CBS 5776 (data not shown), probably
because overliming could not remove acetic and formic
acid which inhibited the fermentability.
In this work, Pichia stipitis CBS 5776 was adapted in
the filtrate with addition of xylose 30g/l step by step, and
at the same time, steam stripping was firstly used to re-
move the volatile compounds such as formic and acetic
acid in the acid hydrolyzate or filtrate, then overliming
was performed to remove more other inhibitors. Xylose
was added up to 45g/l to three kinds of hydrolyzates,
undetoxified hydrolyzate, the hydrolzyate treated with
steam stripping, and the hydrolyzate treated with a com-
bination of steam stripping and overliming. The fer-
mentabilities of the three kinds of hydrolyzates with
yeast Pichia stipitis CBS 5776 were investigated.
2. MATERIALS AND METHODS
2.1. Preparation of Acid Hydrolyzate
Corn stover was obtained from Zhaodong city, Heilong-
jiang Province, China. The main composition of the raw
material was (w/w% of the dry weight): glucan, 40.10;
xylan, 22.30; and lignin, 18.80. The corn stover was
crushed to a granularity of 3 to 5cm, and then steam
treated at 1.8MPa for 5min before explosion. After steam
explosion, 100g (dry weight) exploded corn stover was
washed and filtered three times with 1L distilled water.
The filtrate was then hydrolyzed with dilute sulfuric acid
30g/l at 121℃ for 45min.
2.2. Detoxification of the Acid Hydrolyzate
and the Filtrate
The acid hydrolyzate or the filtrate was heated to boiling
and kept at 100℃. Steam was then put into the bottom of
the vessel with a distributor to perform stripping. Steam
stripping was operated for 15, 30, 45, 60, 90, and 120
min, respectively.
Overliming was carried out by initially adjusting the
pH of the acid hydrolyzate to pH9, 10 and 11, respec-
tively, using solid calcium hydroxide. The samples were
then heated to 40 or 60℃ in a water bath for 90min fol-
lowed by centrifuging at 5000 rpm for 10min. The upper
liquid was collected and stored at 4℃.
A combination of steam stripping and overliming was
also performed for detoxification of the acid hydrolyzate.
The hydrolyzate was firstly detoxified by steam strip-
ping as described above; the sample’s volume was ad-
justed to its original volume by the addition of water.
The sample was then detoxified by overliming proce-
dure.
2.3. Yeast Strain and Media
The yeast Pichia stipitis CBS 5776 was conserved in
Nanjing Forestry University. It was maintained at 4℃ in
a medium containing (g/l): xylose, 20; yeast extract, 5;
peptone, 3; and agar, 20.
Inoculation medium contained 30g/l xylose, 5g/l pep-
tone and 3g/l yeast extract at natural pH. Multiplication
medium was similar to the inoculation medium. The
xylose fermentation medium was described previously
by Yu [25].
Inoculum was prepared in 250ml shaking flask with
100ml medium, and incubated on a rotary shaker at 170
rpm and 30℃ for several batches (24 hours per batch).
When the optical density (OD) of yeast cells reached 10,
the cells were harvested by centrifugation, and the pellet
was inoculated into the fermentation media.
2.4. Adaptable Fermentation
Adaptation medium was prepared by adding 30g/l xy-
lose and the other compositions were described by Yu
[25] in 10%, 20%, 30%, 40% and 50% (v/v) filtrate of
steam exploded corn stover.
The cells from inoculum preparation were firstly in-
oculated into 100 ml of the adaptation fermentation me-
dium of 10% filtrate in 250ml shaking flask at 30℃ on a
rotary shaker at 150 rpm for 22h, then repeated fermen-
tation of 10 % filtrate for two times, finally the cells was
inoculated into 20%, 30%, 40% and 50% filtrate and
fermented for three times, respectively.

J. J. Zhu et al. / Natural Science 1 (2009) 47-54 49
2.5. Fermentation of the Detoxified and
Undetoxified Acid Hydrolyzates
Three acid hydrolyzate preparations, namely the hydro-
lyzate without detoxification, the hydrolyzate treated
with steam stripping only, and the hydrolyzate treated
with a combination of steam stripping and overliming,
were used for fermentation.
The pH of the hydrolyzate preparations was adjusted
to 5.5 by the addition of Ca(OH)2 or H2SO4. The hydro-
lyzate preparations were then centrifuged at 5000 rpm
for 10min; the upper liquids were collected and stored at
4℃. Xylose 45g/l was added to the three hydrolyzate
preparations as the sugar sources. Fermentation was car-
ried out in 250ml shaking flask with 100ml medium, and
incubated on a rotary shaker at 150 rpm and 30℃.
2.6. Analysis
Sugars (cellobiose, glucose, xylose, and arabinose), fer-
mentation products (ethanol, xylitol, glycerol) and in-
hibitors (formic acid, acetic acid, levulinic acid, 5-hy-
droxymethylfurfural, and furfural) were determined by
high performance liquid chromatography (HPLC) using
an Agilent 1100 system and refractive index detector.
Separations were performed on a Bio-rad Aminex HPX-
87H column (300×7.8mm i.d.) at 55℃ using 0.005 mol/l
sulfuric acid as the mobile phase (0.6ml/min). All the
compounds were determined by ESTD methods.
The optical density (OD) of the yeast was measured
spectrophotometrically at 600nm.
Spectrophotometric analysis of the hydrolyzate was
performed using an Amersham Biosciences Ultrosepc
2100 pro UV/visible spectrophotometer. The hydrolyzates
were diluted 100 times for measurements at 280nm.
3. RESULTS AND DISCUSSION
3.1. Compositions of the Filtrate and the
Acid Hydrolyzate from Steam Exploded
Corn Stover
Cellulose, hemicellulose, lignin and extractives in the
corn stover were partially degraded and decomposed
during steam explosion pretreatment [26]. The soluble
degraded products included sugars (glucose, xylose, and
arabinose) and inhibitory compounds (formic acid, acetic
acid, levulinic acid, 5-hydroxymethylfurfural, furfural,
and so on). Soluble oligosaccharides were also found in
the filtrate because they could not be entirely degraded to
monosaccharides during steam explosion, and they could
not be fermented to ethanol by yeasts; therefore 30g/l
sulfuric acid was applied to hydrolyzate the oligosaccha-
rides to monosaccharides such as glucose and xylose at
121℃ for 45min. The contents of the filtrate and the acid
hydrolyzate from steam exploded corn stover were ana-
lyzed with HPLC and were shown in Table 1.
From Table 1, it could be seen that the concentration
of xylose increased more than that of glucose, this indi-
cated that xylooligosaccharides was more easily hydro-
lyzed to monosaccharides than cellooligosaccharides.
After acid hydrolysis of the filtrate, formic acid, acetic
acid and 5-hydroxymethylfurfural were decreased par-
tially; however, levulinic acid and furfural were in-
creased. The changes of inhibitors in the filtrate showed
that a series of degradation reactions were occurred dur-
ing the process of the acid hydrolysis.
3.2. Adaptation Fermentation
Since the sugar contents in the filtrate was too low, 30
g/l xylose was added to the adaptation media containing
10%, 20%, 30%, 40%, and 50% filtrate. Adaptation of
the yeast Pichia stipitis CBS 5776 was achieved in the
adaptation medium. Firstly, Pichia stipitis CBS 5776
was fermented on the adaptation medium of 10 % filtrate
for three times, after each fermentation, the centrifuging
yeast was inoculated in the fresh adaptation medium;
secondly, the yeast was inoculated in the adaptation me-
dium of 20% filtrate for three times. The adaptation se-
quence was continued gradually until the adaptation me-
dium of 50% filtrate. Fig. 1 presented that the changes of
the concentrations of residue xylose, fermentation prod-
uct ethanol, the main fermentation by-product xylitol
adapted fermentation in 10%, 20%, 30%, 40%, and 50%
filtrate for three times by Pichia stipitis CBS 5776.
As shown in Fig. 1, the concentration of residue xy-
lose was increased from 0 in the 10% and 20% filtrate to
19.54g/l (means of triplicate) in the 50% filtrate with the
increasing concentration of the filtrate. The fermentation
product ethanol was firstly a little increased, and then
decreased linearly; the maximum concentration of etha-
nol was 11.59g/l (means of triplicate) in the 20% filtrate.
The fermentation by-product xylitol was decreased
firstly and then increased a little; the maximum concen-
tration of xylitol was 0.95g/l (means of triplicate) in the
10% filtrate.
Table 1. Contents of the filtrate and acid hydrolyzate from
steam exploded corn stover.
Concentration (g/l)
Compounds The filtrate The acid
hydrolyzatea)
Cellobiose 0.33 0.12
Glucose 0.30 1.27
Xylose 0.79 2.13
Arabinose 0.27 0.29
Formic acid 2.07 1.82
Acetic acid 2.03 1.93
Levulinic acid 0.12 0.19
5-hydroxymethylfurfural0.15 0.09
Furfural 0.10 0.18

50 J. J. Zhu et al. / Natural Science 1 (2009) 47-54
0
5
10
15
20
25
10 20 3040 50
the filtrate(%)
xylose（g/l
）
0
2
4
6
8
10
12
14
10 2030 40 50
the filtrate (%)
ethanol （g/l
）
run 1run 2run 3
0
0.5
1
1.5
2
10 20 30 40 50
the filtrate (%)
xylitol（g/l
）
Figure 1. Changes of the concentrations of residue xlose, ethanol and xylitol adapted fermentation in 10 %, 20 %, 30 %, 40 %,
and 50 % filtrate for three times by Pichia stipitis CBS 5776 with the addition of xylose 30g/1.
From the results of the three times of adaptation fer-
mentation in 10% to 50% filtrate by Pichia stipitis CBS
5776, it could be concluded that the fermentability was
decreased gradually with the increasing concentration of
the filtrate, especially in 50% filtrate only 3.55g/l (means
of triplicate) ethanol was produced. So Pichia stipitis
CBS 5776 endured the concentration of inhibitors had
some limitation, only adaptation yeast in the filtrate
could not improved the fermentability significantly.
Therefore, it was necessary to adopt detoxification
methods to detoxify or remove inhibitors in the filtrate in
order to further improvement of fermentation.
3.3. Steam Stripping of the Acid Hydrolyzate
and the Filtrate
Steam stripping was considered as one of the physical
detoxification methods. It only removed the volatile
compounds in the hydrolyzate. The filtrate from steam
exploded corn stover was hydrolyzed at a sulfuric acid
concentration of 30g/l at 121℃ for 45 min as described
above. The resulted acid hydrolyzate was then steam
stripped for 15, 30, 45, 60, 90, and 120 min, respectively.
At the same time, steam stripping of the filtrate was also
investigated. The effects of stripping time on the compo-
sitions of detoxified acid hydrolyzate and the filtrate
were summarized in Tables 2 and 3.
From Tables 2 and 3, it could be seen that the con-
centrations of sugars, such as cellobiose, glucose, xylose
and arabinose, remained essentially the same as expected.
The non-volatile inhibitors, levulinic acid and 5-hy-
droxymethylfurfural, were not removed as well. On the
other hand, the volatile inhibitors, formic acid, acetic
acid and furfural, were removed significantly.
The removal of acetic acid was much higher than that
of formic acid. And the removal of formic acid in the acid
hydrolyzate (1.07g/l) was much more than that in the
filtrate (0.73g/l). The possible reason is that the pKa
value of acetic acid (4.75) is higher than that of formic
acid (3.75). In the acidic condition (pH1-2), acetic acid is
prone to form molecules, which can be removed easily.
While in the filtrate, its pH was 4.00, the formic acid was
existed in ion formation, so it was not easy to remove.
With the stripping time going on, the removal of acetic
acid and formic acid slowed down gradually because the
Table 2. Effect of stripping time on the composition of detoxified the acid hydrolyzate.
Compound (g/l) 0 15 min 30 min 45 min 60 min 90 min 120 min
Cellobiose 0.12 0.11 0.12 0.12 0.12 0.12 0.12
Glucose 1.27 1.27 1.28 1.31 1.32 1.33 1.24
Xylose 2.13 2.10 2.11 2.15 2.17 2.20 2.02
Arabinose 0.29 0.28 0.28 0.29 0.29 0.29 0.28
Formic acid 1.82 1.49 1.34 1.06 0.97 0.81 0.75
Acetic acid 1.93 1.41 1.09 0.70 0.58 0.42 0.37
Levulinic acid 0.19 0.17 0.19 0.20 0.19 0.21 0.21
5-hydroxymethylfurfural 0.09 0.08 0.08 0.08 0.08 0.08 0.06
Furfural 0.18 0.00 0.00 0.00 0.00 0.00 0.00

J. J. Zhu et al. / Natural Science 1 (2009) 47-54 51
Table 3. Effect of stripping time on the composition of detoxified the filtrate of steam exploded corn stover.
Compound (g/l) 0 15 min 30 min 45 min 60 min 90 min 120 min
Cellobiose 0.33 0.32 0.31 0.31 0.30 0.28 0.29
Glucose 0.30 0.29 0.29 0.29 0.28 0.27 0.28
Xylose 0.79 0.80 0.80 0.80 0.76 0.74 0.76
Arabinose 0.27 0.27 0.26 0.26 0.26 0.24 0.25
Formic acid 2.07 1.94 1.82 1.77 1.50 1.40 1.34
Acetic acid 2.03 1.65 1.26 1.04 0.78 0.77 0.46
Levulinic acid 0.12 0.11 0.11 0.11 0.11 0.10 0.11
5-hydroxymethylfurfural 0.15 0.14 0.14 0.14 0.11 0.12 0.13
Furfural 0.10 0.00 0.00 0.00 0.00 0.00 0.00
molecule forms of acetic acid and formic acid were re-
duced with the pH increasing [27].
According to the results of Tables 2 and 3, it was
necessary to hydrolyze the filtrate with dilute sulfuric
acid, because it was not only hydrolyzed the oligosac-
charides to monosaccharides, but also it could be re-
moved more inhibitors in the hydrolyzate. So in this
study, the filtrate was firstly hydrolyzed with 30g/l sul-
furic acid at 121℃ for 45min, and then a steam strip-
ping time of 120min was selected in order to remove
more inhibitors. At this condition, 81% acetic acid and
59% formic acid were removed, while furfural was
stripped off completely.
3.4. Overliming of the Acid Hydrolyzate
The effect of detoxification of the acid hydrolyzate by
overliming on sugars (cellobiose, glucose, xylose, and
arabinose), weak acids (formic, acetic, and levulinic
acid), 5-hydroxymethylfurfural and furfural was exam-
ined. The pH was adjusted to 9, 10, and 11, respectively,
and the temperature was set at 40 and 60 ℃ for each pH
treatment. The overliming time was 90 min [28]. The
results of detoxification were summarized in Table 4.
As shown in Table 4, the concentration of cellobiose,
arabinose, formic acid, acetic acid, levulinic acid and
5-hydroxymethylfurfural were unchanged or changed
slightly when treated with Ca(OH)2 to pH 9-11 at 40 and
60℃. The concentration of xylose and glucose was de-
creased at pH11, especially when higher temperature (60
℃) was applied. Xylose was more destroyed than glu-
cose; this agreed with Martinez’s results [18] in which
pentose sugars were less stable than hexose sugars when
pH was increased from 9 to 11. The concentration of
furfural was reduced by treatment with Ca(OH)2. At
pH11, 41% and 50% furfural were removed at 40 and
60℃, respectively.
The spectrophotometric analyses of different treatments
were also listed in Table 4. Absorbance at 280nm repre-
sents furfural, 5-hydroxymethylfurfural and phenyl ring
absorption band in lignin [22]. Compared with the ab-
sorbance value at 280nm, 1.93, of the untreated acid hy-
drolyzate, the absorbance values of overliming treatment
were decreased with the pH increasing. This indicated that
a part of furfural and phenolics had been removed.
3.5. Detoxification of the Acid Hydrolyzate
with a Combination of Steam Stripping
and Overliming
According to the results of steam stripping and overliming,
the acid hydrolyzate was then detoxified with a two step
method. The acid hydrolyzate was firstly detoxified by
steam stripping for 120min, water was added to maintain
the original value, and then the acid hydrolyzate was sec-
ondly detoxified by overliming at pH11 and 60℃ for 90
min. The composition of the acid hydrolyzate after two
step detoxification was listed in Table 5.
Table 4. The effect of pH and temperature on the overliming detoxification of the acid hydrolyzate.
40℃ 60℃
Compound pH9 pH10 pH11 pH9 pH10 pH11
Cellobiose (g/l) 0.13 0.14 0.12 0.12 0.13 0.11
Glucose (g/l) 1.39 1.39 1.26 1.35 1.33 1.01
Xylose (g/l) 2.23 2.23 2.04 2.16 2.13 1.63
Arabinose (g/l) 0.27 0.27 0.27 0.27 0.27 0.26
Formic acid (g/l) 1.97 1.99 2.04 1.98 1.99 2.04
Acetic acid (g/l) 2.23 2.24 2.26 2.25 2.25 2.28
Levulinic acid (g/l) 0.22 0.22 0.22 0.24 0.24 0.24
5-hydroxymethylfurfural (g/l) 0.07 0.05 0.07 0.09 0.08 0.08
Furfural (g/l) 0.17 0.16 0.10 0.16 0.14 0.08
Absorbance at 280 nm 1.57 1.56 1.35 1.63 1.48 1.36

52 J. J. Zhu et al. / Natural Science 1 (2009) 47-54
Table 5. The composition of acid hydrolyzate detoxified with a combination of steam stripping and overliming.
Compound Before detoxification After steam strippinga) After overlimingb)
Cellobiose (g/l) 0.12 0.07 0.07
Glucose (g/l) 1.27 1.17 0.91
Xylose (g/l) 2.13 1.92 1.23
Arabinose (g/l) 0.29 0.19 0.17
Formic acid (g/l) 1.82 0.59 0.65
Acetic acid (g/l) 1.93 0.35 0.38
Levulinic acid (g/l) 0.19 0.16 0.15
5-hydroxymethylfurfural (g/l) 0.09 0.05 0.05
Furfural (g/l) 0.18 0.01 0.01
Absorbance at 280 nm 1.93 1.11 0.93
a) The time of stripping is 120 min; b) The condition of overliming is at pH11 and 60 ℃ for 90 min.
From Table 5, it could be seen that the concentrations of
formic, acetic acid and furfural were similar to that of
steam stripping alone, indicating that overliming had very
limited effect on the removal of these compounds [18].
The value of absorbance at 280nm was less than that of
overliming alone because steam stripping could remove
some furfural and phenolics. Sugar concentrations after
two step detoxification were lower than both one step
treatments even though the original concentration of each
sugar was at very low level.
3.6. Fermentation of the Detoxified and
Undetoxified Acid Hydrolyzates
The fermentability of three kinds of acid hydrolyzate,
the undetoxified hydrolyzate, the hydrolyzate treated
with steam stripping, and the hydrolyzate treated with a
combination of steam stripping and overliming, had been
investigated. Since the sugar contents in these hydro-
lyzates were too low, 45g/l xylose was added. The yeast
Pichia stipitis CBS 5776 was used as the biocatalyst.
Figs. 2, 3, and 4 illustrated the fermentation courses
including the change profiles of glucose, xylose, the
product ethanol, the by-product xylitol, and the optical
density (OD) of yeast cells.
As shown in Fig. 2, in the fermentation of undetoxi-
fied hydrolyzate, the utilization of sugars were very dif-
ficult. In 36h, only 0.68g/l glucose was consumed; after
48h, the remained 0.30g/l glucose kept untouched. Xy-
lose was utilized very slowly before 48h, indicating a
long period of adaptation was required. When the yeast
adapted to the medium, a rapid consumption of xylose
was observed, and only 0.60g/l xylose was left at 85h.
Ethanol, the main fermentation product, was produced
in accordance with the consumption of sugars. The con-
centration of ethanol reached its highest value of 13.25
g/l at 85 h when xylose was utilized nearly completely
(Fig. 2). After that ethanol concentration decreased, probably
because the yeast used ethanol as the carbon source when the
sugars used out. The trend of the formation of xylitol,
0
10
20
30
40
50
60
0 1224364860728496
time (h)
xylose(g/l), OD
0
5
10
15
20
glucose, xylitol, ethanol (g/l)
OD xylose glucose xylitol ethanol
Figure 2. Changes in the parameters during the fermentation of
undetoxified acid hydrolyzate with the addition of xylose 45g/l.
0
10
20
30
40
50
0 122436486072
time (h)
xylose (g/l), OD
0
5
10
15
20
glucose, xylitol, ethanol (g/l)
OD xylose glucose xylitol ethanol
Figure 3. Changes in the parameters during the fermentation of
acid hydrolyzate detoxified with steam stripping and the addi-
tion of xylose 45g/l.
the main by-product, was similar to that of ethanol. At
72h xylitol concentration reached its peak of 0.67g/l.
The OD was increased constantly from 7.35 to 13.10.

J. J. Zhu et al. / Natural Science 1 (2009) 47-54 53
0
10
20
30
40
50
0 122436486072
time (h)
xylose (g/l), OD
0
5
10
15
20
glucose, xylitol, ethanol (g/l)
OD xylose glucose xylitol ethanol
Figure 4. Changes in the parameters during the fermentation of
acid hydrolyzate detoxified with a combination of steam strip-
ping and overliming, and the addition of xylose 45g/l.
The fermentation of the hydrolyzate treated with steam
stripping was more easily than that of undetoxified hy-
drolyzate as shown in Fig. 3. Glucose was exhausted in
12h. Xylose was consumed rapidly as well; in 36h only
0.38g/l xylose was left. Ethanol reached its highest value
of 13.91g/l at 36h, compared to 85h in Fig. 2. Xylitol
production increased rapidly in 24h, while the OD was
increased constantly from 8.50 to 10.43. Compared with
Fig. 2, the fermentablity of the hydrolyzate treated with
steam stripping was improved significantly.
In the fermentation of the hydrolyzate treated with a
combination of steam stripping and overliming, the
sugar utilization and ethanol production were further
improved compared with Fig. 3. As shown in Fig. 4,
glucose was exhausted in 12h, while xylose was con-
sumed substantially in 24h with a remaining concentra-
tion of 2.28g/l. The peak of ethanol concentration, 15.92
g/l, which was 80.34% of theoretical, appeared at 24h, a
reasonable fermentation time for practical applications.
From the data above (Figs. 2, 3, and 4), it could be
concluded that it is necessary to detoxify the acid hy-
drolyzate in order to ferment the sugars to ethanol.
Steam stripping is an efficient method to detoxify the
hydrolyzate. If combined with overliming, steam strip-
ping could significantly improve the fermentability of
the hydrolyzate in terms of sugar utilization and ethanol
production.
4. CONCLUSIONS
The ability of yeast Pichia stipitis CBS 5776 adapted
gradually on the fermentation medium containing the
filtrate of steam exploded was so limited that detoxifica-
tion methods must be adopted to remove these inhibitors
existed in the filtrate. A combination of steam stripping
and overliming was an effective method to remove the
inhibitors in the steam exploded corn stover prehydro-
lyzate, and could improve the fermentability of Pichia
stipitis CBS 5776. Steam stripping could remove volatile
compounds and at a stripping time of 120min, 81% ace-
tic acid and 59% formic acid were removed while fur-
fural was stripped off completely from the acid hydro-
lyzate. Overliming could reduce the contents of furfural
and phenolics presented in the acid hydrolyzate, how-
ever, sugars, especially pentoses, were also removed
partially. When the acid hydrolyzate detoxified with a
combination of steam stripping for 120min and overlim-
ing at pH11 and 60℃ for 90min, its fermentability was
significantly improved. Xylose was consumed nearly
completely in 24h with an ethanol yield of 15.92g/l,
80.34% of theoretical.
ACKNOWLEDGMENTS
The authors acknowledge the financial supports from National Natural
Science Foundation of China (Grant No.30871992), National High
Technology Research and Development Program of China (Grant No.
2008AA 05Z401), and Natural Science Foundation of Jiangsu Higher
Education of China (Grant No. 06KJA22015).
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