Renewable Energy 54 (2013) 196e200

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Renewable Energy
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Pretreatment of Laminaria japonica for bioethanol production with extremely low

acid concentration
Ji Ye Lee a, Young Soo Kim a, Byung Hwan Um b, KyeongKeun Oh a, *
a
b

Department of Applied Chemical Engineering, Dankook University, Cheonan, Chungnam 330-714, Republic of Korea
Department of Chemical Engineering, Hankyong National University, Anseong, Gyeonggi-do 456-749, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 30 January 2012
Accepted 13 August 2012
Available online 28 August 2012

The conversion of cellulose hydrolysis was investigated in batch reactors from brown macro-algae under
extremely low acid (ELA) pretreatment conditions. The brown macro-algae of Laminaria japonica were
conducted for increasing glucan content as an aqua-biomass feedstock. The investigated ELA pretreatment conditions in this study were as follows; the reaction temperature of 150e180
C, the reaction time
of 5e20 min, and sulfuric acid concentrations of 0.02e0.14%. The maximum glucan content of 29.09%,
which was four-fold higher than that of the raw L. japonica was obtained after the ELA pretreatment
under the optimal condition with sulfuric acid of 0.06% at the temperature of 170
C for 15 min. In
addition, glucan content in solid residue, solid remaining, and the amount of decomposed products in
hydrolyzate through ELA pretreatment of L. japonica were compared with those of hot water treatment.
Signicant enhancement on glucan fraction and enzymatic digestibility of the pretreated L. japonica
could be achieved through the ELA pretreatment.
2012 Elsevier Ltd. All rights reserved.

Keywords:
Extremely low acid (ELA)
Pretreatment
Laminaria japonica
Biomass
Macroalgae

1. Introduction
With rising prices for petroleum and concern for global warming due to the use of fossil fuels the development of renewable fuel
source continues to attract a lot of attention [1e3]. The technology
for conversion of biomass resources to fuels and chemicals, such as
ethanol, has been under development for decades. Over the last
decade, interest has increased in the potential for biofuel as
a means of reducing dependency on fossil fuels and developing
environmentally friendly and renewable energy [4,5]. Biomass
feedstocks include dedicated energy crops, agricultural crops,
forestry residues, aquatic crops, biomass processing residues,
municipal waste, and animal waste. New biomass sources of biofuel
feedstock, such as marine biomass, pre-pulping extract and other
non-food biomass are commonly referred to as next generation (or
sometimes 2nd or 3rd generation, depending on the technology
employed) biofuel feedstocks. In general, they promise higher
productivity, a lower greenhouse gas prole and improved
sustainability performance when compared with 1st generation
feedstocks. Compared to biofuels from agricultural crops, the
amount of land required would be minimal. Marine biomass
comprises of macro and microalgae and both have been proposed

* Corresponding author. Tel.: 82 41 550 3558; fax: 82 41 550 3044.

E-mail address: kkoh@dankook.ac.kr (KyeongKeun Oh).
0960-1481/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.renene.2012.08.025

as potential biofuel feedstocks. Microalgae are potential sources of

bio-oils whilst macroalgae are potential sources of carbohydrates
for fermentation or thermo-chemical based conversions. Previous
research on fuels and energy from kelps has primarily focused on
the production of methane [6], methanol [7] and more recently
ethanol [7e9]. A further study investigated the use of a range of
macroalgae species as combustion fuels [10]. However, marine
biomass is thought to be a promising next generation biofuel
feedstock.
Macroalgae are multicellular, macroscopic algae capable of
generating more kg of dry biomass m2 year1 than fast-growing
terrestrial crops such as sugar cane [11]. The largest growing
macroalgae species are within the phaeophyceae and are termed
kelps. In the Namhae islands which lie off the southern coast of the
Korean Peninsula, the kelps are primarily members of the laminariales order, growing up to 6 m in length. One of the main
considerations for the production of biomass for bioethanol is the
content of carbohydrates of dry feedstock produced. The focus of
this research was therefore on Laminaria japonica, the most prevalent kelp species growing off the Wando, a county in South Jeolla
Province in South Korea, coastline where the samples for this
research were collected.
Currently, commercial enterprises are developing industrial
scale operations with lignocellulose-derived ethanol. Although the
technology has advanced considerably over the past decades,
a much greater understanding of the process fundamentals is

J.Y. Lee et al. / Renewable Energy 54 (2013) 196e200

needed for the new biomass resources. There are many process
congurations possible for converting biomass to ethanol. One of
the well-studied technologies that is currently being commercialized is the use of a dilute acid-catalyzed pretreatment followed by
enzymatic hydrolysis and fermentation to produce ethanol. The
objective of pretreatment is to alter the biomass structure in order
to make the cellulose and non-cellulosic fractions more accessible
to hydrolytic enzymes that can generate fermentable sugars [12].
Hydrothermal pretreatments, including steam explosion, hot water
auto-catalyzed pretreatments, and dilute acid pretreatment, have
been extensively studied in the literature [13e17] and typically
employ hot water or dilute acid to hydrolyze the hemicellulose. For
the case of dilute acid pretreatment, it typically employs 0.4e2%
(w/v) of acid (most commonly sulfuric acid, but possibly nitric
acid, sulfur dioxide, or phosphoric acid) at temperatures of 160e
220
C to recover hemicelluloses and increase the relative
content of cellulose retained on solid phase. However, this kind of
chemical pretreatments is not much performed for making ethanol
with macroalgae feedstocks, which partly accounts for the lack of
literature concerning its pretreatments. There are distinct advantages of using extremely low acid (ELA) conditions for the
pretreatment of cellulosic biomass. Low acidity in the ELA
pretreatment process simplies downstream process such as the
subsequent neutralization and waste treatment as well as signicantly reduces equipment cost [18].
In this study, optimization the hydrothermal pretreatment
conditions for L. japonica, a brown algae, using extremely low acid
(ELA) to signicantly improve glucan content. Evaluation criteria
for optimization of the pretreatment conditions were based on
minimum glucose release in the hydrolyzate and on high glucan
content in the solid residues obtained after ltration of whole
pretreated materials. The sugar fraction of glucan from the solid
phase treated by both ELA and hot water was also evaluated by
enzymatic hydrolysis to evaluate the optimal pretreatment
conditions.
2. Material and methods
2.1. Feedstock collection and preparation
Feedstock of L. japonica (Wando, Korea) was used throughout
this study as a model biomass and provided by the ICPT (Institute of
Cleaner Production Technology, Pukyong National University,
Busan, Korea). This samples were rst dried for 24 h at 45  5
C
and subsequently milled to an average size of 8 mesh (1.40e
2.36 mm) using a blade mill (Blender 7012s, Warning Commercial, CT, USA) and US standard sieve. The screened material was
used directly in pretreatment studies. The moisture content of
milled material was 2.3% based on total weight.

197

treatment time was started to be counted. After the desired reaction time, the reactors were quickly transferred to an ice-water bath
to quench the reaction for 10 min. The tubes were removed from
the water and dried, and the end caps and Teon plugs were
removed. The contents were removed and separated into liquid and
solid fractions by ltration. The conditions for ELA pretreatment
were 150e180
C; 5e20 min; 0.02e0.14% acid concentration. In the
case of pretreatment with hot water, the same liquor to material
ratio was used without any acid charge. The reaction conditions for
hot water pretreatment were 150, 170
C; 20e40 min.
2.3. Glucan analysis
Raw material, pretreated material and hydrolyzate liquor were
analyzed quantitatively for their composition according to NREL
Laboratory Analytical Procedures: NREL/TP-510-42618 (Structural
carbohydrates and lignin) [19]. The Breeze HPLC system (Waters
Co., Milford, MA, USA) used for glucan measurement had a Bio-Rad
Aminex HPX-87H column (300 mm  7.8 mm) and Cation H microguard cartridge (30 mm  4.6 mm), (Bio-Rad Laboratories Inc.,
Hercules, CA). The column was maintained at 60
C, with a 5 mM
H2SO4 eluent at a ow rate of 0.5 mL/min. Sugar peaks were
detected by the RI detector (Waters 2414, Waters Co., Milford, MA,
USA) and identied and quantied by comparison to retention
times of authentic standards.
2.4. Enzymatic hydrolysis experiments
Enzymatic hydrolysis was performed according to NREL standard procedures NREL/TP-510-42629 (Selig et al., 2008) [20].
Commercial cellulase, Celluclast (Novozyme A/S Bagsvaerd,
Denmark) and Novozym 188 (Novozyme A/S Bagsvaerd, Denmark)
were obtained from Korea Institute of Energy Research (KIER,
Korea), and had been purchased from Novozyme Korea Co. The
substrate and control materials were hydrolyzed at the desired
cellulase enzyme loading of 15 FPU/g-glucan and b-glucosidase
loading of 70 pNPGU/g-glucan. Hydrolysis experiments were conducted in 250 mL Erlenmeyer asks with a total working volume of
100 mL while maintaining the glucan concentration of 0.5% (w/v).
The pre-warmed bottle contained 100 mM sodium citrate buffer
(pH 4.8), 80 mg/mL of tetracycline, and 60 mg/mL of cycloheximide
to prevent microbial contamination. The hydrolyzed samples were
boiled to denature the enzyme activity and ltered through a 0.2micron nylon membrane lter at predetermined time periods (i.e.,
0, 1, 3, 6, 9, 12, 18, 24, and 48 h). Triplicate reaction asks were
incubated at 50
C with 120 rpm rotation, and compared to controls
that contained untreated samples.
3. Results and discussion

2.2. Extremely low acid (ELA) and hot water pretreatment

3.1. Effect of pretreatment temperature

The pretreatment experiments were performed in a laboratory

scale stainless steel packed bed reactor with total volume of
15.7 cm3 for each tube. The reactor was loaded with 1.0 g of airdried L. japonica. The residual moisture in the air-dried sample
was accounted for in the determination of the amount of acid
solution to be added, such that a nal ratio of 9 mL liquor per g oven
dry sample was achieved. The tubes were submerged into the rst
bath (molten salt) set at 240
C for rapid preheating. The tubes were
then quickly transferred into the second bath (silicone oil) set at the
desired reaction temperature (150e180
C). This approach allowed
for a rapid heat-up of the non-agitated substrates to the target
temperature in about 1.5 min. When the desired temperature
inside the reactor was reached for second heating bath, the

Fig. 1 presents the soluble glucose and glucan content of

extremely low acid concentration (ELA) pretreatment experiments.
The effects of temperature (ranging from 150 to 180
C) on dilute
H2SO4 (0.10%, w/v) hydrolysis of L. japonica was investigated at the
residence time of 20 min. In order to evaluate the efciency of the
pretreatment temperature the glucan content in the solid phase
and the glucose release in hydrolyzate was determined as
percentage of the dry weight basis of solid remaining after
pretreatment. As can be seen in Fig. 1, the sugar content of the
remaining residue was strongly inuenced by the reaction
temperature employed during ELA pretreatment. In our experiments, the contents selected as four different temperatures, varied
from 21.01 to 32.36%, at the ranging from 150 to 180
C. The

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J.Y. Lee et al. / Renewable Energy 54 (2013) 196e200

order to evaluate the effect of increase of acid dose (up to 0.14% w/

v). Fig. 2 shows the results of an ELA pretreatment on the glucan
content and glucose release as function of acid concentration. The
overall glucan content in the solid residue did slightly increase with
the increase of acid concentration, while the amount of glucose
release kept nearly constant even though the glucan hydrolysis was
enhanced. In Fig. 2, the percent of glucan content calculated was
most inuenced by the relative loss of total mass. For all the
samples treated with either 0.10 or 0.14% (w/v), much of residue
containing glucan was dissolved into the liquid phase even though
the glucan content peaks at the high concentration. Therefore, the
optimal pretreatment condition for this study was selected as 0.06%
H2SO4 at 170
C for 20 min, considering both a relative glucan
content in solid phase and an extent of solid loss due to severity of
pretreatment. On the other hand, the pH of the algal hydrolyzate
obtained from ELA pretreatment was 4.6, which was very close to
the optimal pH (4.8) for the enzymatic hydrolysis of glucan. The
absence of any further needs for neutralization process and/or
waste treatment is important in minimizing the operation cost.
Fig. 1. Effects of reaction temperatures on the glucan content and glucose release using
L. japonica as a substrate (conditions: 0.10% (w/v) H2SO4, 20 min).

maximum glucan content of 32.36% was obtained by hydrolyzing at

0.10% H2SO4 and 170
C for 20 min. Meanwhile, the experimental
glucan content is dramatically decreased at 180
C. In contrast, the
concentration of glucose release in the hydrolyzate increased by the
higher temperature levels (Fig. 1). Interestingly, the formic acid
formation appears to peak at the temperature180
C (data not
shown). This clearly indicates that hexose sugars (glucose) derived
from glucan of L. japonica were further degraded during ELA
pretreatment. However, optimized glucan contents in solid phase
were obtained at the optimal pretreatment condition as 0.10%
H2SO4 at 170
C for 20 min, resulting in a glucose release of 0.7 g/L
in the hydrolyzate and 32.36% glucan content in solid phase.

3.3. Effect of residence time

The effect of the increase of residence time on the pretreatment
of L. japonica was also studied. Fig. 3 shows that an increase in the
residence time in the reactor from 5 to 20 min at a sulfuric acid
concentration of 0.06% also produced a noticeable improvement in
glucan content (from 20.19% to 29.47%). However, the increase of
time (from 10 min to 20 min) resulted in minor increase in the
glucose release (from 0.31 to 0.40%) which was probably due to the
decomposition of the released glucose to degradation products
such as formic acids. Here we see that at the maximum, 27.77% of
the potential glucan content was detected for 0.06% added H2SO4 at
residence time of 15 min in the solid residue after ELA
pretreatment.
3.4. Hot water pretreatment results

The extremely low H2SO4 pretreatment of L. japonica was

carried out for the predetermined condition (170
C, 15 min) in

In order to evaluate the efciency of the pretreatment process,

hot water pretreatment experiments carried out at the temperature
of 150 and 170
C and the ranging of reaction time from 5 to 20 min.
Fig. 4 shows the hot water hydrolysis conditions and glucan content

Fig. 2. Effects of ELA concentrations on the glucan content and glucose release using
L. japonica as a substrate (conditions: 170
C, 15 min).

Fig. 3. Effects of reaction times on the glucan content and glucose release using
L. japonica as a substrate (conditions: 0.06% (w/v) H2SO4, 170
C).

3.2. Effect of ELA concentration

J.Y. Lee et al. / Renewable Energy 54 (2013) 196e200

Fig. 4. The glucan content and glucose release of L. japonica treated by hot water
(conditions: 0.06% (w/v) H2SO4, 150e170
C, 20e40 min).

in solid phase and glucose release in hydrolyzate after the hydrolysis experiments. The effects of temperature (150 and 170
C) on
glucose and glucan yields for various reaction times (ranging from
20 to 40 min) were evaluated. The glucan contents depicted as bar
in solid phase are shown in Fig. 4, and ranged from 14.59% to
24.83%, depending on the pretreatment conditions. The glucan
content achieved in this study are in the slightly different range as
ELA pretreatment studied. In comparison to glucan content treated
with ELA, glucan content treated with hot water was found to lower
content from all pretreatment tested at the same pretreatment
conditions. The higher potential glucan contents on the remaining
residue in the present study are achieved through ELA pretreatment than that of hot water pretreatment. For the glucose release in
the hydrolyzate, tests on L. japonica showed similar results, with
ELA and hot water pretreatments ranging from 1.25 to 4.85% and
from 1.25 to 5.53 of their sugar concentration respectively. Meanwhile, we found that the amount of formic acid decomposed
product for hot water pretreatment is slightly higher than that of
ELA pretreatment (data not shown). In our experiments, it can be
found that ELA pretreatment is an effective pretreatment for
L. japonica because the liberation of glucose can be minimized from
the glucan through an extremely lower acidic hydrolysis maximizing the remaining glucan for enzymatic digestion. Under the
hydrolysis conditions used approximately 79.20% and 82.04% of the
mass of the material was extracted. These values and the percent of
sugar remaining in the solid residue are shown in Table 1.
3.5. Enzymatic hydrolysis results
Enzymatic hydrolysis experiments were conducted to select the
best pretreatment condition to yield maximum enzymatic digestibility for further research. Fig. 5 presents results of a batch enzyme
Table 1
Percent solid remaining, glucan content, and enzymatic digestibility of ELA pretreated L. japonica and hot water treated L. japonica.

Untreated
ELA pretreated
Hot water treated

Solid
remaining (%)

Glucan
content (%)

Enzymatic
digestibility (%)

100
20.8
18.0

6.9
29.1
24.8

34.91
83.37
81.08

199

Fig. 5. Enzymatic digestibility of L. japonica treated by ELA and hot water.

hydrolysis carried out on six different nutrient broth solutions: two

control solution containing untreated and a-cellulose diluted with
buffer solution, a solution containing ELA treated sample diluted
with buffer solution after washing, two solutions containing ELA
treated samples diluted with DI water and hydrolyzate without
washing process respectively, the last solution containing hot water
treated sample diluted with buffer. The highest digestibility of the
ELA treated sample diluted with buffer after washing is 83.37% after
24 h at 15 FPU/g-glucan in our experiments, while the digestibility
of the sample diluted by DI water and hydrolyzate was 80.77% and
74.38% at the same condition. On the basis of different dilution
tested, as expected, it was found that the glucose digestibility for
the mixture of ELA treated sample: DI water is higher than that of
ELA treated sample: hydrolyzate mixture. Through the dilution test,
we found that glucan digestibility is affected by the presence of
hydrolyzate in the enzyme broth. This inhibition may be related to
decompose products (i.e. formic and acetic acids) dissolved in the
hydrolyzate. In addition, the mode of inhibition of the acid on
enzyme hydrolysis may be via reduction of the pH below the
optimal range, resulting in a decrease in glucose release.
4. Conclusion
Different levels of ELA pretreatment of marine biomass can be
accomplished over the range of reactor temperatures, acid
concentration, and residence times investigated. This level of
pretreatment can have a profound in impact on the fermentable
sugar content in the solid residue of marine biomass. This initial
study has demonstrated that extremely low sulfuric acid pretreatment is a useful approach for L. japonica to the release of glucose
minimize from the glucan through a lower acidic hydrolysis
maximizing the remaining glucan for enzymatic digestion in solid
residue. This project has demonstrated that the relative fraction of
glucan fermented to ethanol in the L. japonica can be increased
efciently by ELA H2SO4- catalyzed hydrothermal pretreatment.
The maximum glucan content of 29.09%, which was four-fold
higher than that of the raw L. japonica was obtained after the ELA
pretreatment under the condition with sulfuric acid of 0.06% at the
temperature of 170
C for 15 min. In our experiments, it could be
found that the ELA pretreatment is a promising way to achieve the
advanced purpose of macro-algae pretreatment. Specically, the
process advantage of the ELA pretreatment is that it does not

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J.Y. Lee et al. / Renewable Energy 54 (2013) 196e200

require a neutralization option for the enzymatic hydrolysis. In

addition, this study can serve as a step for the further development
of some empirical models for marine biomass to predict and optimize the experimental data such as potential glucan content which
will be used for ethanol fermentation as feedstock.
Acknowledgments
This work was nancially supported by the Ministry for Food,
Agriculture, Forestry and Fisheries (20111001212-00).
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