Bioprocess Biosyst Eng (2013) 36:713–719
DOI 10.1007/s00449-013-0895-5
ORIGINAL PAPER
Bioethanol production from brown seaweed, Undaria pinnatifida,
using NaCl acclimated yeast
YuKyeong Cho • Hyejin Kim • Sung-Koo Kim
Received: 23 December 2012 / Accepted: 10 January 2013 / Published online: 30 January 2013
! Springer-Verlag Berlin Heidelberg 2013
Abstract Strain improvement of Pichia angophorae
KCTC 17574 was successfully carried out for bioethanol
fermentation of seaweed slurry with high salt concentra-
tion. P. angophorae KCTC 17574 was cultured under
increasing salinity from five practical salinity unit (psu, %)
to as high as 100 psu for 723 h. The seaweed, Undaria
pinnatifida (sea mustard, Miyuk), was fermented to pro-
duce bioethanol using high-salt acclimated yeast. The
pretreatment of U. pinnatifida was optimized using thermal
acid hydrolysis to obtain a high monosaccharide yield.
Optimal pretreatment conditions of 75 mM H2SO4 and
13 % (w/v) slurry at 121 "C for 60 min were determined
using response surface methodology. A maximum mono-
saccharide content of 28.65 g/L and the viscosity of
33.19 cP were obtained. The yeasts cultured under various
salinity concentrations were collected and inoculated to the
pretreated seaweed slurry after the neutralization using 5 N
NaOH. The pretreated slurry was fermented with the
inoculation of 0.1 g dcw/L of P. angophorae KCTC 17574
strain obtained at 90 psu. The maximum ethanol concen-
tration of 9.42 g/L with 27 % yield of theoretical case of
ethanol production from total carbohydrate of U. pinnati-
fida was obtained.
Keywords Seaweed ! Undaria pinnatifida ! Bioethanol !
Continuous stirred tank reactor (CSTR)
Y. Cho ! H. Kim ! S.-K. Kim (&)
Department of Biotechnology, Pukyong National University,
Busan 608-737, Korea
e-mail: skkim@pknu.ac.kr
Introduction
The depletion of fossil fuels and the increasing problem of
CO2 emissions have led to seek alternative sources for
biomasses such as corn stover, lignocellulosic biomass,
forest products residue and seaweeds [1, 2]. Biomass is a
renewable energy resource providing transportation fuels
such as bioethanol or biodiesel [2, 3]. Bioethanol has been
regarded as a major substitute to replace the liquid fossil
fuels among them [4].
Bioethanol has been produced from agricultural feed-
stock and lignocellulosic biomass in many countries [3, 4].
Sugarcane has been already used as the biomass for ethanol
production on a commercial scale in Brazil [4]. However,
agricultural feedstock has caused moral problems and price
instability [2]. Lignocellulosic biomass is a second gener-
ation biomass which is cheap and available in large
quantities [5]. However, lignin combined with cellulose is
difficult to degrade the cellulose. Therefore, biomass is a
major factor to be considered for feasible bioethanol
production.
The seaweed has been considered as a third generation
biomass for bioethanol production [6]. The seaweed has
high productivity per unit area per year, and there is no
competition with food crops. The seaweed can be grown
rapidly compared to land-based biomass. Undaria pin-
natifida (sea mustard, Miyuk), Saccharina japonica (sea
tangle, Dasima) and Porphyra tenera (purple laver, Pare)
are the main species grown in the Korean coasts. Among
them, U. pinnatifida has spread worldwide growing rapidly
with the maximum length of 3 m [7].
The carbohydrate contents of seaweed are in a range of
30–70 %. It depends on the species and culture conditions.
The brown seaweed can be a promising substitute due to
well-developed cultivation method and mass production in
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Korea. Carbohydrates in brown seaweed consist of algi-
nate, laminaran, fucoidan and mannitol [8]. Main compo-
nent of brown seaweed is alginate ranging up to 50 % of
the carbohydrate [9]. Degradation of these polysaccharides
is slow and requires specific enzymes. Therefore, it is
essential to develop suitable pretreatment methods based
on biomass properties. Thermal acid hydrolysis was
evaluated to improve the yield of saccharification in
U. pinnatifida.
Salt concentration is also important for the ethanol
production from seaweeds because salt could act as an
inhibitor, resulting in reductions of cell growth and fer-
mentation yield when salt concentration is over certain
concentration. Salt-tolerant yeasts have been used to pro-
duce bioethanol and various polyols (glycerol, i-erythritol,
arabitol, mannitol) in a number of industrial applications
[10]. Debaryomyces hansenii and Zygosaccharomyces
rouxii have been used as the tolerant yeasts at high salt
concentrations [10, 11]. However, most yeasts used for
typical ethanol production do not have the ability to tol-
erate a high salt condition. Thus, yeast strains at high
salinity were acclimated to produce ethanol from seaweed,
U. pinnatifida using continuous stirred tank reactor
(CSTR).
Using U. pinnatifida as the biomass for ethanol pro-
duction, this study was carried out to increase the mono-
saccharide contents and improve the fermentation yield by
optimizing thermal acid hydrolysis conditions and yeast
strain improvement at high salt concentrations.
Materials and methods
Raw materials
The seaweed, U. pinnatifida, harvested in February 2010
and discarded as waste after the seaweed packaging pro-
cess, was obtained from Gijang Local Products Co., Ltd. in
Busan, Korea. The seaweed was dried with sunlight or hot-
air and ground by hammer mill. The ground seaweed was
sieved by 200 mesh and used for analysis of crude protein,
crude lipid, carbohydrate and ash of seaweed by AOAC
method [12]. Crude lipid was determined by using an ether
extraction procedure (Soxtec System 1046, foss, Hoganas,
Sweden) after freeze-drying for 12 h.
Thermal acid hydrolysis
Seaweed with different sulfuric acid concentrations and
slurry contents of solid were pretreated at the ranges of
37.5–187.6 mM and 10–20 % (w/v) in 250 mL Erlen-
meyer flask with a working volume of 100 mL. The slurry
was then autoclaved for 15–60 min at 121 "C.
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Bioprocess Biosyst Eng (2013) 36:713–719
Monosaccharide content, viscosity and salinity of each
sample were measured.
Yeast inoculum and CSTR culture
Yeast, Pichia angophorae KCTC 17574, was obtained
from Korean collection of type culture (KCTC) and prepared
on YPD agar plate. YPD agar plate containing 20 g/L yeast
extract, 10 g/L peptone, 20 g/L dextrose and 5 % (w/v)
agar was used for the yeast cultivation. A single colony was
purified and the seed culture was prepared with working
volume of 5 mL in liquid medium. We inoculated 5 % (v/v)
from the seed culture to the second culture, and it was
prepared in 250 mL Erlenmeyer flask with a working
volume 100 mL and incubated in a shaking incubator at
30 "C with 220 rpm for 12 h. Pre-cultured yeast strain was
incubated in CSTR with a working volume of 2.5 L of
YPD medium containing salts to improve salt-tolerant
strain. The salt concentrations of medium in the reactor
were increased by addition of sodium chloride. The initial
salinity was 50 psu. The medium salinities were increased
at the rate of 5 psu up to 100 psu, and each cultivation
medium was maintained for 72 h. The high-salt acclimated
yeast was cultured in 250 mL fresh YPD medium adding
salt after 72 h of cultivation. Cultured yeast strain was
sampled to measure the colony forming unit (CFU) and pH.
Fermentation with pretreated hydrolysate
Fermentation was carried out in 250-mL Erlenmeyer flask
with a working volume of 100 mL. The seaweed slurry
pretreated by thermal acid hydrolysis at optimal conditions
was neutralized to pH 7 by 5 N NaOH. A colony of
improved yeast strains was obtained and cultivated in YPD
medium containing each salinity for 24 h. The seed culture
was prepared from pre-cultured inoculation at each salinity
medium. And then yeast strains of 0.1 g dcw/L, P. ango-
phorae KCTC 17574, were added to the pretreated sea-
weed slurry that has the same salinity. The medium was
incubated on 30 ± 2 "C for 72 h and sampled to determine
the concentration of ethanol and acetic acid.
Analysis
Cell growth was measured by UV–vis spectrophotometer at
OD600 and converted to dry cell weight using the standard
curve. The slurry of thermal acid hydrolysis was analyzed
to determine the concentrations of monosaccharide and
organic acid by high performance liquid chromatography
(HPLC) (Agilent 1100 Series, Agilent Inc., USA) equipped
with refractive index detector (RID). Bio-rad Aminex
HPX-87H column (300.0 9 7.8 mm) was used with
degassed 5 mM sulfuric acid at the flow rate of 0.6 mL/min
Bioprocess Biosyst Eng (2013) 36:713–719 715

and the temperature of 65 "C. The samples of fermentation
were prepared by centrifugation for 8 min at 12,000 rpm,
and the supernatant was treated with 0.2 lm filter prior to
analysis. Viscosity was measured by Brookfield viscometer
(BROOKFIELD DV-III Rheometer v3.1, Brookfield Eng.
Inc., USA) equipped with spindle No. ULA, SC4-18 and
SC4-34 at temperature of 30 "C. Salinity was measured by
salinometer (Salinity Refractometer, ATAGO Inc., Japan).
Response surface methodology (RSM)
The influences of three variables were determined by a
response surface methodology (RSM) using SAS 9.1 (SAS
Institute, Cary, NC, USA). The experiments were designed
by central composite design (CCD) based on 23 factors.
Seventeen experiments were set up according to Table 1
and analyzed at each range of three variables.
Results and discussion
The composition of Undaria pinnatifida
The composition of U. pinnatifida was analyzed and shown
in Table 2. U. pinnatifida contained 48.5 % of carbohy-
drate, 18.2 % of crude protein, 1.8 % of crude lipid,
28.0 % of crude ash and 3.5 % of crude fiber. Carbohy-
drate content of U. pinnatifida used in this study was
52.0 % carbohydrate including crude fiber as cellulose on
dry solid basis.
Thermal acid hydrolysis
Thermal acid hydrolysis was evaluated on the basis of the
effects of three factors: acid concentration, treatment time
and solid content as variables for the degradation of car-
bohydrate in U. pinnatifida. Acid concentration, treatment
time and solid content were coded as X1, X2 and X3 for the
analysis by RSM (response surface methodology) on the
yield of monosaccharide and viscosity which were coded to
Yy and Yv as responses, respectively.
All regression coefficients in quadratic model were
significant (P \ 0.05), and all variables have important
effects on yield of monosaccharide. F value (11.25) and
P value (\0.01) demonstrated that the model was statisti-
cally significant. Regression coefficient (R2) indicated that
92.5 % of variation could be explained in the model.
The saccharification of carbohydrate to monosaccharide
was related to acid concentration and treatment time as
shown in Fig. 1a. The effects of acid concentration at
121 "C were evaluated. The yield of monosaccharide
increased from 29 to 42 % with the increase of acid con-
centrations from 7.5 up to 93.0 mM and decreased with
further increases of acid concentrations over 93.0 mM.
High acid concentrations generally showed better results of
releasing high amounts of monosaccharide [13]. However,

Table 1 The central composite

Run Independent variables Response
design employed for three
independent variables Sulfuric acid Treatment time Solid content Yield of Viscosity
concentration (mM) (min) (%) monosaccharide (%) (cP)
X1 X2 X3 Yx Yy

1 -1 (37.5) -1 (30) -1 (10) 15.5 48.8

2 -1 (37.5) -1 (30) 1 (16) 16.9 8,686.2
3 -1 (37.5) 1 (60) -1 (10) 33.3 59.49
4 -1 (37.5) 1 (60) 1 (16) 31.4 3,143.3
5 1 (112.5) -1 (30) -1 (10) 19.2 12.8
6 1 (112.5) -1 (30) 1 (16) 25.8 29.4
7 1 (112.5) 1 (60) -1 (10) 24.2 15.3
8 1 (112.5) 1 (60) 1 (16) 26.4 35.39
9 -a (22) 0 (45) 0 (13) 26.8 4,239.1
10 a (128) 0 (45) 0 (13) 18.4 18.2
11 0 (75) -a (24) 0 (13) 20.3 57.9
12 0 (75) a (66) 0 (13) 38.8 40.2
13 0 (75) 0 (45) -a (9) 31.5 14.6
14 0 (75) 0 (45) a (17) 28.8 2,339.5
15 0 (75) 0 (45) 0 (13) 34.9 35.7
16 0 (75) 0 (45) 0 (13) 34.8 31.5
17 0 (75) 0 (45) 0 (13) 34.5 32.4

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716 Bioprocess Biosyst Eng (2013) 36:713–719

Table 2 General composition of U. pinnatifida on dry basis
Component
Crude protein
Crude lipid
Crude carbohydrate
Crude ash
Crude fiber
high acid concentration caused sugar decomposition, thus,
formed into fermentation inhibitors such as furfural and
hydroxyl methyl furfural (HMF) [3, 13]. These inhibitors
prevent the ethanol fermentation process as well as cell
growth. Therefore, proper acid concentration should be
applied to minimize inhibitor production for the fermen-
tation. In this study, furfural and HMF were not detected
with increasing acid concentration, and a maximum
monosaccharide content of 28.8 g/L was achieved with
75 mM sulfuric acid.
The effects of treatment times were also studied. Fig-
ure 1a shows that the increase of treatment time increased
the yield of monosaccharide production from 29 to 42 %
with the increase of treatment time from 30 to 60 min at
the acid concentration of 75 mM, respectively. High
monosaccharide content was obtained by extending the
treatment time; however, the energy consumption was high
considering the yield of monosaccharide [14].
Contents of slurry also changed with various acid con-
centration and treatment time. The viscosity was dependent
on the solid content of slurry as shown in Fig. 1b. High
solid contents of slurry produced high monosaccharide
contents by the conversion of polysaccharide to monosac-
charide [14]. However, high solid contents of slurry caused
high viscosity, which makes it difficult to handle the
Dry basis (%, w/w)
medium [15]. Therefore, proper solid contents should be
applied.
18.2 Although this model did not explain the effects and
1.8 interactions among variables, it showed a tendency
48.5 between factors. This quadratic model showed a statistical
28.0 significance of (\0.0001) with a high R2 value of 0.929.
3.5 The optimal conditions considering these variables were
determined with 13 % (w/v) slurry and 75 mM sulfuric
acid at 121 "C for 60 min. A maximum monosaccharide
content of 28.8 g/L and 42 % of conversion from total
carbohydrate was obtained with viscosity of 33.19 cP.
Effect of salinity on cell viability
One of the problems encountered in seaweed was high
concentrations of NaCl due to its origin of sea water [16].
Additional salts produced during thermal acid hydrolysis
due to the neutralization cause difficulty in the ethanol
production process. If this salt concentration is higher than
the optimal condition of yeasts in the fermentation of
seaweed, salts inhibit the fermentation process with low
ethanol production [17].
In order to maintain the cell growth and fermentation
activity of yeast at high salt concentrations, continuous
stirred tank reactor (CSTR) culture was used to change the
metabolic activities of P. angophorae strain. The accli-
mation of the yeast, P. angophorae KCTC 17574, to the
salinity of 50–100 psu was carried out, and the results are
shown in Fig. 2. The initial salinity of YPD medium was
50 psu, and the colony forming unit (CFU) of 1014–1015
CFU/mL was obtained at the medium salinities from 50 to
70 psu. However, the CFU decreased with the increase of

(a) 50
(b) 14000
30 min
30 min
Yield of monosaccharide (%)

45 min 12000
40 60 min 45 min
60 min
10000
Viscosity (cP)

30
8000

6000
20
4000

10 2000

0
20
0 18 200
20
18
Slu 16
)
(mM
16 rry 14 150
200
Slu
rry
14
12 150 (%
)
12 100
rat ion
(%) 10 100
(mM) 10
ent
tration
50
onc
8 50
oncen 8
dc
Acid c Aci

Fig. 1 Response surface on a effects of acid concentration (mM), slurry contents (%) and treatment time (min) on the yield of reducing sugar
contents, b effects of acid concentration (mM), slurry contents (%) and treatment time (min) on viscosity

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Bioprocess Biosyst Eng (2013) 36:713–719
Fig. 2 Cell viability of acclimated strains on Pichia angophorae
KCTC 17574 at various salt concentrations with CSTR
8 9
salinity. The cell numbers of 10 –10 CFU/mL were
obtained at the salinity of 75 psu, and the colony shape was
also changed due to the osmotic pressure. Although the cell
number decreased at high salt concentrations up to
100 psu, cells obtained from CSTR culture grew well in the
YPD medium containing high salt concentration up to
100 psu. These yeast strains changed the metabolic system
and possessed salt tolerance, allowing them to survive the
high salt concentration. This indicates that these salt
acclimated yeast strains are salt-tolerant.
Ethanol fermentation
The slurries pretreated by optimal thermal acid hydrolysis
condition were used for the fermentation media. Salt
acclimated yeast strains of P. angophorae KCTC 17574
from various salt concentrations were used to produce
ethanol from seaweed, U. pinnatifida. P. angophorae pro-
duced ethanol using fermentable monosaccharide of
D-mannitol, D-glucose and D-xylose [18]. The major com-
ponents in the brown seaweed, U. pinnatifida, are alginate,
laminaran and mannitol. Monosaccharides produced from
seaweed components can be used by P. angophorae for the
ethanol production.
In this study, yeast strains cultured at the salinity range
of 80–100 psu were used for the ethanol fermentation
because the salinity of medium becomes 90 psu after the
pretreatment and neutralization. The fermentation was
carried out by the addition of 0.1 g dcw/L of yeast strain
dispersion to the pretreated slurry. The fermentation med-
ium was incubated at 30 ± 2 "C with 220 rpm for 72 h.
The result of ethanol production at various salinities is
shown in Fig. 3. The ethanol concentration of 9.42 g/L was
obtained with strain cultured at 90 psu. And the ethanol
concentration of 5.84 g/L was observed in the strain cul-
tured at salinity of 85 psu. Other yeast strains cultured at
80, 95, 100 psu produced ethanol concentration of less than
717
12
80 psu
85 psu
10 90 psu
95 psu
100 psu
8 Wild-type
EtOH (g/L)
6
0
0 20 40 60 80
Time (hrs)
Fig. 3 Ethanol production of U. pinnatifida with acclimated strains
cultured at 80 psu (open triangle), 85 psu (filled square), 90 psu
(filled circle), 95 psu (filled triangle) and 100 psu (open square)
including wild-type strain (open circle)
2 g/L. These results show that matched salinities between
acclimated yeast and fermentation media are required for
the production of ethanol. Ethanol concentration of 2 g/L
was produced by fermentation with wild-type P. ango-
phorae strain, and similar results were obtained by
unmatched salinities between yeast and seaweed broth as
shown in Fig. 3.
The highest ethanol concentration was observed using
improved strain cultured at 90 psu, and the time course of
ethanol production is shown in Fig. 4. The ethanol con-
centration of 9.42 g/L was obtained with 27.3 % of theo-
retical yield of total carbohydrate at 36 h of fermentation.
The fast ethanol production rate by improved strain at high
salt concentration was obtained within the first 36 h, and
then ethanol concentration decreased slowly. Two days are
sufficient to produce ethanol, and then the ethanol degraded
into other products. The initial pH of fermentation medium
was 6.5, and the pH decreased to 4.54 due to the by-
products from ethanol fermentation. After reaching the
highest ethanol concentration, acetic acid was produced
during the fermentation process. Most of the by-products of
conversion of sugars to ethanol are glycerol, succinic acid
and acetic acid [19]. Acetic acid concentration increased by
the decrease of ethanol concentration, and the maximum
acetic acid concentration of 4.46 g/L was produced at 72 h
of incubation as shown in Fig. 4. This result shows that
ethanol was oxidized and turned into acetic acid probably
due to acetic acid production bacteria after 36 h of incu-
bation. It has been reported that acetic acid is produced in
the lignocellulosic biomass due to the degradation of dif-
ferent components which constitute the biomass and can
act as potential fermentation inhibitors if acetic acid con-
centration exceeds 6 g/L [20, 21]. The result shows that
acetic acid concentration was increased to 4.46 g/L at the
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718 Bioprocess Biosyst Eng (2013) 36:713–719

Fig. 4 Ethanol and acetic acid 12 14 100

production, pH and salinity Salinity (psu)
(psu) of U. pinnatifida with salt EtOH (g/L) 12
10 Acetic acid (g/L)
acclimated yeast strain, Pichia 80
pH
angophorae KCTC 17574, 10

Acetic acid (g/L)

obtained by CSTR culture at 8

Salinity (psu)
EtOH (g/L)
90 psu with optimal 60
8
pretreatment condition: ethanol

pH
6
concentration (filled circle), 6
Salinity (filled square), pH 40
(filled triangle down) and acetic 4
4
acid concentration (open
20
triangle) 2
2

0 0 0
0 20 40 60 80
Time (hrs)

end of the fermentation. Acetic acid concentration less than References

2 g/L did not affect the ethanol production, however, eth-
anol production was decreased under the acetic acid con-
centration over 2 g/L in our experiments.
Our studies show that the highly salt-tolerant yeast
strain, P. angophorae KCTC 17574 produced by CSTR
culture, can produce high concentration of ethanol com-
pared to that of wild-type strain. Studies on wet-milling
using wet macroalgae are carried out to reduce the unit
operation like drying which requires energy and cost. Also,
further studies on the improvement of yeast and carbohy-
drate degradation strains [22] are essential for the utiliza-
tion of total carbohydrate in macroalgae.
Conclusion
The seaweed, Undaria pinnatifida, is a promising substitute
biomass for the bioethanol production due to its rapid
growth and high productivity. For the pretreatment by
thermal acid hydrolysis, three variables, acid concentra-
tions, treatment times and the solid contents of slurry
affecting the monosaccharide yield and viscosity were
studied using the response surface method. The optimal
thermal acid hydrolysis conditions were 75 mM sulfuric
acid and 13 % (w/v) slurry at 121 "C for 60 min. High
monosaccharide contents of 28.42 g/L with 42.5 % of
theoretical yield were obtained.
Salt-tolerant yeast strain at 90 psu, Pichia angophorae
KCTC 17574 was obtained by CSTR culture, and ethanol
concentration of 9.42 g/L with 27.3 % of theoretical yield
from total carbohydrate of U. pinnatifida was produced by
fermentation.
Acknowledgments The research was supported by a grant from
Marine Biotechnology Program Funded by Ministry of Land,
Transport and Maritime Affairs of Korean Government.
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