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Bioresource Technology 140 (2013) 421425

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Biotransformation of 5-hydroxymethylfurfural (HMF) by


Scheffersomyces stipitis during ethanol fermentation of hydrolysate
of the seaweed Gelidium amansii
Chae Hun Ra a, Gwi-Taek Jeong a, Myung Kyo Shin b, Sung-Koo Kim a,
a
b

Department of Biotechnology, Pukyong National University, Busan 608-737, Republic of Korea


Biolsystems Co. Ltd., Gwan-ri 430, Doyang-eub, Goheung-gun, Jeonnam, Republic of Korea

h i g h l i g h t s
 Thermal acid hydrolysis and enzymatic saccharication resulted in 43.5 g/L sugars.
 Ethanol concentration of 20.5 g/L was obtained using S. stipitis KCTC 7228.
 Glucose uptake increased at HMF concentrations <5 g/L in the fermentation medium.
 HMF was converted to 2,5-bis-hydroxymethylfuran by S. stipitis KCTC 7228.

a r t i c l e

i n f o

Article history:
Received 31 January 2013
Received in revised form 29 April 2013
Accepted 29 April 2013
Available online 9 May 2013
Keywords:
Ethanol fermentation
HMF
Biotransformation
Gelidium amansii
Scheffersomyces stipitis

a b s t r a c t
The seaweed, Gelidium amansii, was fermented to produce bioethanol. Optimal pretreatment condition
was determined as 94 mM H2SO4 and 10% (w/v) seaweed slurry at 121 C for 60 min. The mono sugars
of 43.5 g/L with 57.4% of conversion from total carbohydrate of 75.8 g/L with G. amansii slurry 100 g
dcw/L were obtained by thermal acid hydrolysis pretreatment and enzymatic saccharication. G. amansii
hydrolysate was used as the substrate for ethanol production by separate hydrolysis and fermentation
(SHF). The ethanol concentration of 20.5 g/L was produced by Scheffersomyces stipitis KCTC 7228.
The effect of HMF on ethanol production by S. stipitis KCTC 7228 was evaluated and 5-hydroxymethylfurfural (HMF) was converted to 2,5-bis-hydroxymethylfuran. The accumulated 2,5-bis-hydroxymethylfuran in the medium did not affect galactose and glucose uptakes and ethanol production.
Biotransformation of HMF to less inhibitory compounds by S. stipitis KCTC 7228 could enhance overall
fermentation yields of seaweed hydrolysates to ethanol.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Marine macroalgae (seaweed) has emerged as an alternative
and promising feedstock to produce a myriad of renewable fuels
(Park et al., 2012). Use of seaweed as an energy feedstock for production of biodiesel, bioethanol, biogas, and biohydrogen has been
investigated (Adams et al., 2009; stgaard et al., 1993). Seaweed
has a faster growth rate, lower land usage, higher CO2 absorption
and uptake rate, no need for fertilizers and no competition for food
or freshwater resources compared to lignocellulosic biomass,
(Singh et al., 2011). The red seaweed Gelidium amansii has a high
content of easily degradable carbohydrates, making it a potential
substrate for the production of liquid fuels. The carbohydrates in
red seaweed comprise a neutral polymer (agarose) and a sulfate
polysaccharide (agaropectin). Therefore, galactose and glucose

Corresponding author. Tel.: +82 52 629 5868; fax: + 82 51 629 5863.


E-mail address: skkim@pknu.ac.kr (S.-K. Kim).
0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.04.122

are obtained by agarose and agaropectin hydrolysis (Park et al.,


2012).
Various pretreatment techniques have been introduced to enhance the overall hydrolysis yield, and can be categorized into
physical, chemical, biological, enzymatic or a combination (Agbor
et al., 2011). Dilute acid hydrolysis is commonly used to prepare
seaweed hydrolysates for enzymatic saccharication and fermentation for economic reasons. However, the red seaweed G. amansii
contains numerous inhibitory compounds, such as 5-hydroxymethylfurfural (HMF), which are generated by thermal acid hydrolysis pretreatment. Furfural is derived from the dehydration of
pentose and HMF is formed from the dehydration of hexoses during sugar degradation by thermal acid (Boopathy et al., 1993; Larsson et al., 1999). These compounds damage microorganisms by
reducing enzymatic and biological activities, breaking down DNA,
and inhibiting protein and RNA synthesis (Modig et al., 2002; Sanchez and Bautista, 1998).
Additional treatments are needed to solve the problems represented by these inhibitory compounds and to facilitate the

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C.H. Ra et al. / Bioresource Technology 140 (2013) 421425

fermentation process; these include physical, chemical, and biochemical treatments. These additional steps add cost and complexity to the process and generate additional waste products.
However, few microbial transformations of furfural and HMF are
known (Boopathy et al., 1993). The yeast Scheffersomyces stipitis
KCTC 7228 converts HMF into a chemical compound presumed
to be 2,5-bis-hydroxymethylfuran (Liu et al., 2004). Therefore, biotransformation of HMF by S. stipitis KCTC 7228 is a promising alternative that avoids the need for separate detoxication steps.
The objective of this study was to optimize the pretreatment
conditions for ethanol production and to demonstrate the transformation of HMF to the HMF conversion product (2,5-bis-hydroxymethylfuran) using S. stipitis KCTC 7228.

Ethanol fermentation was performed with 100 mL of seaweed


hydrolysate in a 250 mL Erlenmeyer ask under semi-anaerobic
conditions. Enzymatic saccharication and nal neutralization to
pH 5.5 were carried out after thermal acid hydrolysis. A yeast seed
culture was grown at 30 C, 120 rpm for 24 h in 5 mL YPD medium
containing 20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose and
transferred to a 250 mL Erlenmeyer ask containing 100 mL of seaweed hydrolysate. The seaweed hydrolysates were fermented at
30 C, and 200 rpm with S. stipitis KCTC 7228. Samples were taken
periodically for determination of ethanol, residual sugars, and HMF
concentrations and stored at 20 C prior to analysis.

2. Methods

2.4. Analytical methods

2.1. Yeast strains and seaweed preparation

The glucose, galactose, HMF, HMF conversion product, levulinic


acid, and ethanol concentrations in samples were determined by
HPLC (Agilent 1100 Series, Agilent. Inc., Santa Clara, CA, USA)
equipped with refractive index detector. The Bio-Rad Aminex
HPX-87H column (300.0  7.8 mm) was maintained at 65 C, and
samples were eluted with 5 mM H2SO4 at 0.6 mL/min. HMF and
2,5-bis-hydroxymethylfuran were conrmed by analysis using
HPLC equipped with a C18-lm column (Bondpack) and a dualwavelength UV detector. The samples were used with 20-fold dilution of hydrolysate solution. The mobile phase was 0.3% acetic acid
and methanol with a linear gradient program at a ow rate of
0.4 mL/min (Matejicek et al., 2003). The activities of cellulase and
b-glucosidase were determined according to the procedure described in Mandels et al. (1976) and Kubicek (1982).

S. stipitis KCTC 7228 (Kurtzman and Suzuki, 2010) was obtained


from the Korean Collection for Type Cultures (KCTC), Biological Resource Center (Daejeon, Korea). G. amansii (product of Morocco)
was obtained from the Biolsystems Co., Ltd., Goheung-gun, Korea.
The seaweed was dried in sunlight or hot air and then ground in
a hammer mill. The seaweed powder was separated with a 200mesh sieve prior to pretreatment. The composition and proximate
analyses of G. amanssi were carried out by the Feed and Foods
Nutrition Research Center at Pukyong National University in Korea,
according to the AOAC method (AOAC, 1995).
2.2. Pretreatment of G. amansii seaweed
Optimization of thermal acid hydrolysis conditions was carried
out with an 814% (w/v) seaweed slurry, 56168 mM H2SO4, and
45240 min autoclaving time. Thermal acid hydrolysis was performed using 100 mL seaweed slurry and H2SO4 in a 250 mL Erlenmeyer ask. Seaweed slurry hydrolysates were then neutralized to
pH 5.0 with 5 N NaOH. Saccharication was conducted by adding
0.024 U/mL of Viscozyme L (1.2 FBG/mL, beta-glucanase, Novozymes, Bagsvaerd, Denmark) and 0.168 U/mL Celluclast 1.5 L
(8.4 EGU/mL, endo-glucanase, Novozymes) to 100 mL of seaweed
slurry. Samples were taken periodically and centrifuged. The
supernatants were analyzed for mono sugars using HPLC. The efciencies of thermal acid hydrolysis pretreatment, enzymatic saccharication and ethanol yield were calculated as follows:

EP % DSg =TC  100


in which Ep is efciency of thermal acid hydrolysis pretreatment
(%), DSg is galactose increase (g/L) during thermal acid hydrolysis
pretreatment, TC is total carbohydrate (g/L) in pretreated G.
amansii.

Es % DSg =C  100

in which Es is efciency of enzymatic saccharication (%), DSg is glucose increase (g/L) during enzymatic saccharication after the pretreatment, C is cellulose content (g/L) in pretreated G. amansii.

Y EtOH g=g EtOHmax =Sugarini


in which YEtOH is ethanol yield (g/g), [EtOH]max is maximum ethanol
concentration achieved during fermentation (g/L), [Sugar]ini is total
initial fermentable sugar (galactose + glucose) concentration at onset fermentation (g/L).

2.3. Fermentation of the seaweed hydrolysate to ethanol

3. Results and discussion


3.1. Optimization of the pretreatment conditions
The composition of G. amansii was analyzed by the AOAC method and found to contain 58.4% carbohydrate, 17.4% crude ber,
18.7% crude protein, 0.7% crude lipid, and 4.8% crude ash and etc.
The galactose contents from the agarose and agaropectin by
thermal acid hydrolysis treatments were evaluated. The optimal
conditions in terms of slurry content and H2SO4 concentration for
various thermal hydrolysis times are shown in Fig. 1. The determination of optimal conditions was carried out with slurry content of
814% (w/v), H2SO4 concentrations of 56281 mM and 45
240 min thermal hydrolysis. As shown in Fig. 1a, galactose concentration increased with increasing slurry content, and the maximum
galactose concentration at 14% (w/v) slurry content was 28.5 g/L
with Ep of 35%. However, increasing the slurry content during thermal acid hydrolysis resulted in the decrease of Ep from 40% to 35%.
Levels of levulinic acid and HMF inhibitors increased with increasing slurry content. Therefore, 10% (w/v) of seaweed content with Ep
of 40% was selected for ethanol production.
The thermal acid hydrolysis of carbohydrate to galactose was
dependent on acid concentration and thermal acid hydrolysis
duration as shown in Fig. 1b and c. The effects of acid concentration
were evaluated at 10% (w/v) slurry content, 121 C for 60 min.
Redding et al. (2010) reported high acid concentrations showed
the release of high amounts of mono sugar. However, Ep after treatment with 131281 mM H2SO4 was not greater than that with
94 mM H2SO4 at 10% (w/v) slurry content (Fig. 1b). Therefore,
94 mM H2SO4 was selected as the optimal acid concentration with
Ep of 40% for thermal acid hydrolysis pretreatment.
Efciency of pretreatments at 10% (w/v) slurry and 94 mM
H2SO4 with thermal acid hydrolysis duration are shown in
Fig. 1c. Galactose concentration increased slightly with increasing

C.H. Ra et al. / Bioresource Technology 140 (2013) 421425

423

Glucose contents from cellulose in G. amansii by enzymatic saccharication were determined. The effect of single and mixed enzyme treatments on glucose release of G. amansii hydrolysate
with 10% (w/v) slurry after thermal acid hydrolysis was evaluated
as shown in Fig. 2. The initial glucose was 3.0 g/L after thermal acid
hydrolysis pretreatment. For the hydrolysis of cellulose,
0.168 EGU/mL of Celluclast 1.5 L and 0.024 FBG/mL of Viscozyme
L were used to 100 g/L of seaweed slurry after a thermal acid
hydrolysis at pH 5.0, 40 C for 48 h. The glucose conversion between single and mixed enzyme treatment was observed. Single
treatments of Viscozyme L and Celluclast 1.5 L were 2.4 g/L with
Es of 13% and 10.5 g/L with Es of 60%, respectively. Enzyme treatment of Celluclast 1.5 L to G. amansii hydrolysate was preferable
than that of Viscozyme L. Among those treatment methods, the
mixed enzyme treatment to G. amansii hydrolysate showed synergistic effect and maximum efciency of enzymatic saccharication,
Es. The maximum Es was 96% with 16.7 g/L of glucose from crude
ber of 17.4 g/L from 100 g dcw/L G. amansii slurry. The optimum
enzyme reaction time was 48 h, and further increase in reaction
time up to 72 h had no signicant effect on Es. The total mono sugar concentrations of 43.5 g/L and 57.4% of conversion from total
carbohydrate of 75.8 g/L from 100 g dcw/L G. amansii slurry were
obtained.
3.2. Separate hydrolysis and fermentation (SHF)
The yeasts applied for ethanol production have a very narrow
substrate range (Horn et al., 2000). The hydrolysate from G. amansii
showed different concentrations of galactose and glucose, however
a successful utilization of mixed sugars was carried out for high
ethanol yield (YEtOH) using S. stipitis KCTC 7228. The cell concentration of original seed culture was determined as 5.4 g dcw/L. Five
mL of seed culture were inoculated to 100 mL of G. amansii hydrolysate medium. SHF was carried out by inoculating 0.27 g dcw/L S.
stipitis KCTC 7228 as nal cell concentration into the thermal acid
and enzyme hydrolyzed G. amansii slurry. As shown in Fig. 3, the
impact of HMF on ethanol production was assessed with 8, 10,
and 12% (w/v) slurry contents at 30 C and 200 rpm under semianaerobic conditions. At a seaweed slurry concentration of 8%
(w/v, Fig. 3a), ethanol concentration increased rapidly from 36 to
60 h when HMF decreased to near zero. After glucose was exhausted, galactose was consumed due to the preference for

Fig. 1. Effect of thermal acid hydrolysis using various seaweed contents, acid
concentrations, and thermal hydrolysis durations on efciency of pretreatments:
(a) G. amansii slurry content, (b) H2SO4 concentration and (c) thermal hydrolysis
time.

thermal acid hydrolysis duration. Thermal acid hydrolysis durations of 45240 min at 121 C showed similar Ep of 4042% and resulted in HMF concentrations of 5.811.8 g/L. Due to the inhibitory
effect of HMF, a 60 min thermal acid hydrolysis was used in subsequent experiments. Therefore, the optimal thermal acid hydrolysis
conditions were: 10% (w/v) slurry content, 94 mM H2SO4, at 121 C
for 60 min. From these results, the fermentation inhibitors such as
HMF and organic acids were generated by thermal acid hydrolysis
as pretreatment of red seaweed G. amansii. These inhibitory compounds could stress S. stipitis KCTC 7228, thus, the efcient utilization of sugars was reduced and ethanol production was decreased
as previously reported by Mussatto and Roberto (2004).

Fig. 2. Effect of single and mixed enzyme treatments on glucose release of G.


amansii hydrolysate at 10% (w/v) slurry after thermal acid hydrolysis at pH 5.0,
40 C for 48 h. The initial glucose was 3.0 g/L after thermal acid hydrolysis
pretreatment.

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C.H. Ra et al. / Bioresource Technology 140 (2013) 421425

Fig. 4. Conversion of HMF to the conversion product 2,5-bis-hydroxymethylfuran


by S. stipitis KCTC 7228 Hydrolysates of 10% (w/v) slurry were fermented at 30 C
and 200 rpm under semi-anaerobic conditions.

3.3. Biotransformation of HMF during ethanol production

Fig. 3. Ethanol production from G. amansii (product of Morocco) by separate


hydrolysis and fermentation (SHF) using S. stipitis KCTC 7228 with (a) 8% (w/v), (b)
10% (w/v) and (c) 12% (w/v) of seaweed slurry contents through thermal acid
hydrolysis and enzyme treatment.

glucose. The ethanol concentration and yield with 8% (w/v) slurry


content showed 16.7 g/L with YEtOH of 0.46 at 72 h. The ethanol
productions with 10% (w/v, Fig. 3b) and 12% (w/v, Fig. 3c) slurry
contents were 20.5 g/L with YEtOH of 0.47 at 96 h and 21.0 g/L with
YEtOH of 0.43 at 156 h, respectively. Ethanol yields (YEtOH) of this
study were higher than that of the yeast Brettanomyces custersii
KCTC 18154P with YEtOH of 0.38 (Park et al., 2012). Thus, S. stipitis
KCTC 7228 could be suitable yeast for the fermentation of G. amansii hydrolysate. Fig. 3c shows use of seaweed slurry over 10% (w/v)
increased lag phase before ethanol production due to the presence
of HMF. These results indicate that increased HMF concentration
inhibited both galactose or glucose uptakes and ethanol production
by the yeast. However, the presence of HMF below 5 g/L in the fermentation broth resulted in rapid glucose uptake and ethanol production as shown in Fig. 3(a and b). Slurry contents of 8% (w/v) and
10% (w/v) showed similar ethanol yield (YEtOH). However, the ethanol concentration was low in 8% (w/v) slurry content, thus 10%
(w/v) slurry was selected as optimal slurry content for the production of bioethanol.

Ethanol production was carried out using 10% seaweed hydrolysate by SHF. The ability of S. stipitis KCTC 7228 to transform HMF to
the HMF conversion product was assessed to better understand the
decrease in HMF during ethanol production by HPLC equipped
with UV detector. The HMF peak was detected in 0 h of fermentation broth at UV 282 nm. And then, HMF peak was not detected at
UV 282 nm after 60 h of fermentation broth. However, a new HMF
conversion peak was detected in 60 h of fermentation broth at UV
222 nm (data not shown).
The conversion mechanism of furfural to furfural alcohol has
been well established (Morimoto and Murakami, 1967; Palmqvist
et al., 1999; Taherzadeh et al., 2000). These reports indicated that
HMF was converted to another compound by yeast and interpreted
it as HMF alcohol since HMF has a structure similar to furfural
(Nemirovskii et al., 1989). A similar UV/Vis spectrum was reported
by Boopathy et al. (1993) during investigation of Klebsiella pneumonia and other enteric bacteria for HMF metabolism. Liu et al.
(2004) later identied HMF conversion product with maximum
absorbance at 222 nm as 2,5-bis-hydroxymethylfuran. Fig. 4 shows
the decrease in HMF with concomitant increase in 2,5-bis-hydroxymethylfuran during ethanol fermentation by S. stipitis KCTC 7228.
The biotransformation uses aldehyde as an electron accepter, simply converting HMF to HMF conversion product by 2e reduction
(Belay, 1997). As the formation of 2,5-bis-hydroxymethylfuran as
conversion product from HMF, Figs. 3 and 4 show that 2,5-bishydroxymethylfuran in the medium did not affect galactose and
glucose uptakes and ethanol production. Therefore, S. stipitis KCTC
7228 could be converted HMF into a conversion product as 2,5bis-hydroxymethylfuran. To decrease HMF concentration, thus
the ethanol fermentation could be carried out without any damage
to yeast.
4. Conclusions
A maximum mono sugar concentration of 43.5 g/L and 57.4% of
conversion from total carbohydrate of 75.8 g/L with G. amansii
slurry 100 g dcw/L were obtained from thermal acid hydrolysis
as pretreatment and enzymatic hydrolysis. The ethanol concentration was 20.5 g/L with YEtOH of 0.47 at 96 h by S. stipitis KCTC 7228.
The HMF conversion product detected by HPLC at UV 222 nm was
presumed to be 2,5-bis-hydroxymethylfuran. The fermentation
proles by S. stipitis KCTC 7228 provided a basis for the ethanol

C.H. Ra et al. / Bioresource Technology 140 (2013) 421425

fermentation and production of HMF conversion product, 2,5bis-hydroxymethylfuran.


Acknowledgement
This work was supported by a Research Grant of Pukyong
National University (2013 year).
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