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Fermentation of Oligosaccharides in Softwood Spent Sulite Liquor to Ethanol by Pichia stipitis
Keishi Tanifuji
a,b
, Shiho Takahashi
b
, Kevin Shiell
c
, M. Sarwar Jahan
b
, Yonghao Ni
b
, Hiroshi Ohi
a
a
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8572,Japan oi.hiroshi.gmu.tsukuba.ac.jp
b
Department of Chemical Engineering and Limerick Pulp and Paper Centre, University of New Brunswick, Fredericton, NB, E3B 5A3,Canada, yonghaounb.ca
c
Bioreinery Technology Scale-up Centre, New Brunswick Community college, Grand Falls, NB, E3Y 3W3, Canada Kevin.Shiellgnb.ca
ABSTRACT
Pichia stipitis is one of the feasible strains that can produce ethanol from oligosaccharides. Spent sulite
liquor SSL from softwood contains monosaccharides, oligosaccharides and fermentation inhibitory compounds, such as acetic acid, furfural and sulite ion. The objective of this study is to develop an effective method for the
removal of inhibitory compounds without decrease of oligosaccharides concentration in SSL, so that the ethanol production from oligosaccharides in SSL is maximized. Firstly, the effect of inhibitory compounds on ethanol
fermentation from cellobiose by P. stipitis was investigated. The presence of more than 5 gL of acetic acid in media totally inhibited ethanol production from cellobiose and no ethanol was produced. At 1 gL of acetic acid,
2.6 gL of ethanol was obtained after 40 h fermentation; which was compared to 2.9 gL of ethanol production from the control cellobiose; 13.9 gL, no acetic acid, 24h fermentation. Secondly, the removal of acetic acid
in the SSL by the combination of CaO and the strong base anion exchange resin treatment was studied. The acetic acid concentration of SSL was decreased from 5.8 to 0.9 gL by the strong base anion exchange resin
treatment for 2 min followed by the CaO treatment. The decrease of oligosaccharides concentration in softwood SSL was marginal. Finally, the improvement of ethanol production from oligosaccharides in the SSL by using the
combination of CaO and strong base anion exchange resin treatment was studied. The ethanol was obtained 1.3 gL from the SSL treated by the combined methods, while 6.5 gL of total oligosaccharides were consumed. No
ethanol was obtained from the untreated SSL.
Keywords: ethanol, fermentation, Pichia stipitis, spent sulite liquor, oligosaccharide
Introduction
Sulite cooking is an established process in the pulp and paper industry. In a previous study, when
acid sulite cooking was conducted at 150°C for 1-4 h at pH 1.4, gluco-oligosaccharide and manno-
oligosaccharide were obtained in softwood spent sulite liquor SSL[1].
Also, a
softwood sulite pulp with kappa number of 127 had a high resolution ratio
71.4 by enzyme treatment, which was higher than that of soda-anthraquinone and kraft pulps [1,2].
Several sulite pulp mills have utilized SSL for ethanol production [3]. Baker’s yeast Saccharomyces
cerevisiae can ferment hexose, however, it can’t ferment oligosaccharides and pentose. Further
hydrolysis steps such as acid or enzyme treatments
are required for ethanol fermentation in order to utilize oligosaccharide in SSL, which would
require additional stages and
equipments. A xylose fermentable strain Pichia stipitis has the
genes for seven b-glucosidases, one b -mannosidase
and one endoxylanase, thus facilitating oligosaccharide utilization [4, 5]. It has been reported that P. stipitis
can produce ethanol from cellobiose [6, 7] and xylan [8]. Parekh and Wayman reported that P. stipitis CBS
5776 gave 10.3 gL of ethanol from 25 gL cellobiose within 48 h [7]. Lee et al. reported that P. stipitis CBS
5775 can lead to the production of 2 gL of ethanol from 2 larch wood xylan solution [8].
The SSL also contains furfural, acetic acid and sulfate ion [9], which inhibit the ethanol production
from monosaccharides with microorganisms [10]. P. stipitis is more sensitive to inhibitory compounds than
S. cerevisiae [11, 12]. However, the effect of inhibitory compounds on oligosaccharide fermentation has not
been well understood. Moreover, it is necessary to remove inhibitory compounds from SSL to achieve
effective ethanol fermentation.
We have studied that the combined CaO and strong base ion exchange resin treatments of hardwood
SSL to remove inhibitory compounds [13]. We have concluded that the combined treatment of CaO and
two-stage strong base ion exchange resin treatments in a relatively short time can achieve the selective
removal of acetic acid. The two-stage ion exchange resin treatment removed acetic acid by approximately
90 from the CaO-treated SSL.
The objective of this study is to apply the same concept to a softwood SSL, that is, to develop an
effective method to improve the ethanol production from softwood SSL. This was achieved by the
removal of inhibitory compounds without decrease
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of oligosaccharides in SSL so that the ethanol production from oligosaccharides in softwood SSL is
maximized. Firstly, the effect of inhibitory compounds on ethanol fermentation from cellobiose by using P.
stipitis
was clariied. Secondly, the removal of acetic acid in softwood SSL by the combination of CaO
and the strong base anion exchange resin treatment was studied. Thirdly, the improvement of ethanol
production from oligosaccharides in softwood SSL by using the combination of CaO and strong base anion
exchange resin treatment was studied.
Experimental Procedure SSL Sample
The softwood based SSL was obtained from a pulp mill in Eastern Canada. The chemical composition of
SSL is shown in Table 1.
Ion Exchange Resins
The strong base ion exchange resin Diaion PA408 Cl
-
form was obtained from Mitsubishi Chemical and used for the experiments. The OH
-
form of PA408 was prepared as follows; the Cl
-
form resin was soaked in 2 N NaOH for 30 min, then iltered. This treatment was
repeated 5 times, subsequently the resin was washed by distilled water 5 times. The OH
-
form of PA408 was kept in distilled water to prevent oxidation and
denaturation.
Combined Treatment of CaO and Strong Base Ion Exchange Resin Treatments
The CaO treatment of SSL was conducted as follows. The pH of hardwood SSL was adjusted to
10.5 with 100 gL of CaO slurry and then treated at 70°C for 15 min in water bath [14, 15]. The mixture
was separated with iltration and iltrate was used for further experiments. The CaO-treated SSLs with or
without neutralization with CO
2
were treated with the OH
-
form of PA408 for up to 5 min at 30°C with 150 rpm.
Microorganism
P. stipitis CBS 6054 was obtained from USDA. Stock cultures were kept on YPDX ager plate which contained
yeast extract, 10 gL; peptone, 20 gL; D-glucose, 10 gL; D-xylose, 20 gL and agar, 10 gL [16].
Inoculum Preparation
Inoculum was prepared by transferring a loopful of colonies from agar plate into 50 ml of media which
contained peptone, 3.5 gL; yeast extract, 3 gL; KH
2
PO
4
, 2 gL; MgSO
4
, 1 gL; NH
4 2
SO
4
, 1 gL and xylose, 80 gL, pH 5.0 in 125 ml of Erlenmeyer lask. Incubation
was conducted at 30°C and 150 rpm for 48 h. The yeast was collected by centrifugation at 5000 rpm for 5 min
and washed with sterile distilled water twice [17].
Fermentation
In the model fermentation, cellobiose 15 gL solution in the presence of various acetic acid, furfural
and SO
2
concentrations 0.01, 0.1, 1, 5 and 10 gL were prepared. In the fermentation of SSL, the untreated,
CaO-treated and ion exchange resin-treated SSLs were used. These SSL samples were concentrated to make
the total oligosaccarides concentration of 15 gL. The peptone, yeast extract and ions, which were the same
amount as those used for the inoculum preparation, were added to samples. About 1 gL of dry cell weight
was used. All of the fermentation experiments were conducted at 30°C with 225 rpm [13].
Analytical Methods
Monosaccharaides concentrations were measured using an ion chromatography unit equipped with
CarboPac® PA1 column Dionex-300, Dionex Corporation, USA and a pulsed amperometric detector
PAD [18]. Oligosaccharides were hydrolyzed by 4 H
2
SO
4
at 121 ˚C for 1 h and then measured by ion chromatography unit. Oligosaccharides concentration
was calculated by the subtract sugar concentration before hydrolysis from that after hydrolysis. The
furfural, acetic acid and ethanol concentrations were determined by
1
H-NMR method as described in the literature [19]. The lignosulfonate concentration was
determined by the absorbance at 205 nm with UV spectrometry [20]. A calibration curve was prepared
by using commercial lignosulfonate. The cellobiose concentration was determined using an HPLC
equipped with RI detector Shimadzu. Separations were performed at 65˚C on Rezex ROA-organic Acid
H
+
column phenomenex. Injection volume was 20 mL. The mobile phase was 5 mM H
2
SO
4
. The low rate was 0.6 mLmin.
Results and Discussion Effect of Inhibitory Compounds on Ethanol
Production from Cellobiose by Using P. stipitis Ethanol and cellobiose concentrations during
fermentation in the presence of inhibitory compounds are shown in Fig. 1 and Fig. 2, respectively. The
maximum ethanol concentration from the control without inhibitory compounds during fermentation
was 2.9 gL. It was found that the presence of more than 5 gL of acetic acid in media totally inhibited
ethanol production from cellobiose and no ethanol was produced Fig. 1 A.
A 1 gL of acetic acid
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inhibited ethanol production until 24 h, but after 40 h of fermentation, 2.6 gL of ethanol was obtained,
which was almost the same as that of the control. At this time, the amount of cellobiose consumption
was increased signiicantly from 24 h to 40 h Fig. 2 A. The above results indicated that the inhibition
of cellobiose fermentation caused by acetic acid was negligible in the late stage of fermentation. Glucose
was not found from all of fermented samples.
The maximum ethanol concentrations with 1 gL of furfural and 0.1 gL of sulite ion were 1.9 gL
and 1.3 gL, respectively Fig. 1 B and 1 C. Both of which still showed strong inhibition even at the late
stage of fermentation 40 h. Also, cellobiose in 1 gL
of furfural and 0.1 gL of sulite ion model solutions were not completely assimilated at the late stage of
fermentation and its concentrations were 2.2 gL and 3.3 gL, respectively; while all of cellobiose in the
control was assimilated Fig. 2 B and 2 C.
Fig. 3 shows the change of acetic acid and furfural concentrations during fermentation. The acetic acid
concentration was reduced after 16 h fermentation. On the other hand, furfural concentration only changed
marginally. Implied here is that P. stipitis consumed acetic acid during fermentation. This is the reason why
the inhibitory effect of acetic acid was reduced in the late stage of fermentation.
In addition, the ethanol production from 1 gL acetic acid model solution was higher than that from
1gL furfural solution Fig. 1 A and 1 B, and less furfural was assimilated than acetic acid Fig. 3. Even,
1gL of sulite ion inhibited ethanol production Fig. 1 C. These results indicate that the negative effect of
acetic acid on the inhibition of cellobiose fermentation was less than that of furfural or sulite ion.
Fig. 3. Change of Acetic Acid and Furfural Concentrations during Cellobiose Fermentation with P.
stipitis. Fig. 1. Ethanol Production from Cellobiose by P. stipitis in The Presence of Inhibitory Compounds: A Acetic
Acid; B Furfural; C Sulite Ion
Fig. 2. Cellobiose Consumption during Fermentation in The Presence of Inhibitory Compounds: A Acetic Acid; B
Furfural; C Sulite Ion
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Removal of Inhibitory Compounds from Softwood SSL by Combination of CaO and Ion Exchange
Resin Treatments
The removal of acetic acid from the SSL by the combination of CaO and ion exchange resin
treatments are shown in Fig. 4. In the previous study, the combined CaO and two-stage ion exchange resin
treatments removed approximately 90 of acetic acid from a hardwood SSL, and 29 of total sugars were
also removed in the same process [13]. In this study, the acetic acid concentration in softwood SSL was
decreased from 5.8 gL to 0.9 gL by the combined CaO treatment and one-stage ion exchange treatment
for 2 min. The total monosaccharides concentration was also decreased by this treatment 1.8 gL,
however, the total oligosaccharides concentration did not change much. It was reported that the OH
-
form of strong base anion exchange resin can adsorb monosaccharides and consequently decompose them
[21-23]. Oligosaccharides could be hardly adsorbed to ion exchange resin, thus their decomposition was
minimum.
The CaO treatment of SSL was applied for the recovery of lignosulfonate in pulp mills [14]. The
sulfate and sulite ions can also be removed from the SSL by the CaO treatment [14, 15]. Other studies have
also shown that furfural in the pre-hydrolyzates or SSL can be removed by the lime treatment [16, 24]. It
was revealed that inhibitory compounds in SSL could be removed by the combined CaO and ion exchange
resin treatments without decreasing oligosaccharides.
Enhanced Ethanol
Production from Oligosaccharides in softwood SSL by Combined
CaO and Ion Exchange Resin Treatments Table 1 shows the chemical composition of
SSL samples before and after fermentation. The fermentation results are shown in Fig. 5. When
the fermentation with P.stipitis was conducted for 24 h, 1.3 gL of ethanol was obtained in the SSL
treated by the combined CaO and ion exchange resin treatments. All of monosaccharides were
consumed at 16 h fermentation. In addition, 3.3 gL mannooligosaccharides, 1.9 gL glucooligosaccharides
and 0.1 gL of xylooligosaccharides were consumed Table 1. P. stipitis has the genes for
seven
b-glucosidases, one b-mannosidase and one endoxylanase [4, 5]. Oligosaccharides were hydrolyzed
by these enzymes and consequently consumed by the strain. Several reports have shown that P. stipites could
produce ethanol from cellobiose [6, 7] and xylan [8], however limited results were available on utilization
of mannooligosaccharides by P.stipitis. The present
results conirmed that P. stipitis has an ability to metabolize mannooligosaccharide. As shown in Fig.
5, further oligosaccharides consumption from the SSL Fig. 4. Time Dependence of Removal of Acetic Acid and Sugars in Softwood SSL by Strong Base Ion Exchange
Resin PA408 Treatment: A Acetic Acid; B Total Monosaccarides; C Total Oligosaccharides Table 1. Chemical Composition of SSLs Samples Before and After Fermentation
Monosaccharides gL Oligosaccharides
Glu Man
Xyl Ara
Gal Gul
Man Xyl
Ara Gal
Acetic acid Untreated
1.0 3.3
0.8 0.7
3.9 9.1
0.3 1.5
5.2 CaO treated
0.9 2.3
0.5 0.5
3.9 9.1
0.4 1.4
4.9 CaO and ion exchange resin
0.7 1.8
0.4 0.4
3.9 9.1
0.4 1.4
1.1 After fermentation for 24 h
2.0 5.8
0.2 1.3
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treated with combined CaO and ion exchange resin did not occurred after 16 h fermentation.
b-glucosidases and
b -mannosidase, which are produced by P.stipitis have
high speciicities to hydrolyze cellobiose and mannobiose.
The ethanol concentration from the CaO treated SSL after 40 h fermentation was 0.5 gL, and no
ethanol was obtained from the untreated SSL. It was revealed that the combined CaO and ion exchange
treatments of SSL are effective on improving ethanol production from oligosaccharides in softwood SSL.
Conclusions
It was found that acetic acid in the softwood sulite spent liquor SSL can be removed by the combined
CaO and ion exchange resin treatments so that the acetic acid concentration in the SSL was decreased
from 5.8 gL to 0.9 gL by the combined method. The combined method was effective on improving
ethanol production from oligosaccharides in SSL with P. stipitis. 38 of total oligosaccharides .SSL
treated by the combined method, while no ethanol was obtained from the untreated SSL. From the result
of model experiments using cellobiose solution, 2.6 gL of ethanol was obtained with 1 gL of acetic acid
present, while 2.9 gL of ethanol was produced from the control with no acetic acid present. The acetic
acid concentration was decreased during the P. stipitis fermentation, thus, the inhibitory effect of acetic acid
was lowered in the late stage of fermentation. The negative effect of acetic acid on the inhibition of
cellobiose fermentation was less than that of furfural
or sulite ion.
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