J Appl Phycol
DOI 10.1007/s10811-013-0068-6
Bioethanol production from the hydrolysate of Palmaria
palmata using sulfuric acid and fermentation
with brewers yeast
Siti Mutripah & Maria Dyah Nur Meinita &
Ji-Young Kang & Gwi-Taek Jeong & AB Susanto &
Romanus Edy Prabowo & Yong-Ki Hong
Received: 5 February 2013 / Revised: 4 June 2013 / Accepted: 5 June 2013
# Springer Science+Business Media Dordrecht 2013
Abstract Seaweeds, particularly species of red macroalgae,
are promising resources for bioethanol production because
of their exceptionally high carbohydrate content. Of 20 sea-
weeds evaluated, Palmaria palmata (Rhodymenia palmata)
contained the highest carbohydrate content (469.8 mg g1
seaweed) with a carrageenan content of 354 mg g1 seaweed.
Such a high carrageenan content makes the high-volume
production of bioethanol feasible. Acid hydrolysis of P.
palmata in 0.4 M H2SO4 at 125 C for 25 min released
27 mg of glucose, 218.4 mg of reducing sugars, and
127.6 mg of galactose per gram of seaweed. Ethanol fermen-
tation of these hydrolysis products using an inoculum con-
centration of 1.5 mg mL1 at 30 C and 72 h in a shaking
incubator at 130 rpm yielded 17.3 mg of ethanol per gram of
seaweed.
Keywords Seaweed . Palmaria palmata . Rhodophyta
carrageenan . Acid hydrolysis . Bioethanol . Fermentation
S. Mutripah : R. E. Prabowo
Department of Biology, Jenderal Soedirman University,
Purwokerto, Indonesia
M. D. N. Meinita (*)
Department of Fisheries and Marine Science,
Jenderal Soedirman University, Purwokerto, Indonesia
e-mail: maria.meinita@unsoed.ac.id
J.<Y. Kang : G.<T. Jeong : Y.<K. Hong
Department of Biotechnology, Pukyong National University,
Busan 608-737, South Korea
ABSusanto
Susanto
A.
Department of Fisheries and Marine Science,
Diponegoro University, Semarang, Indonesia
Introduction
The use of nonrenewable fossil fuels is unsustainable and
contributes to the accumulation of greenhouse gasses in the
atmosphere (Edwards et al. 2003; Hossain et al. 2008; Singh
et al. 2010). The necessity of renewable and substitute fuels is
increasing with the depletion of fossil fuel supplies. In con-
trast, ethanol is an energy source produced by the fermentation
of renewable resources such as lignocellulosics and marine
resources (Corro and Ayala 2008; Kang et al. 2012; Wyman
1994). Currently, ethanol is commercially produced from
crops such as corn (Zhao and Xia 2010) and sugarcane (Wu
et al. 2011). Recently, commercial ethanol production from
lignocellulosic resources has been demonstrated (Geddes et al.
2011; Sims et al. 2010).
Nevertheless, limited terrestrial resources have diverted at-
tention to potential marine resources for bioethanol production.
Toward this goal, seaweeds have been identified as potential
candidates for ethanol production (Yanagisawa et al. 2011).
The use of seaweed to produce bioethanol does not affect food
stocks since seaweed is not a major human foodstuff. Seaweed
also contains considerably high levels of complex sugars or
polysaccharides (Chynoweth 2002) and nonlignins, and ex-
hibits a high biomass growth rate (Adams et al. 2009; Meinita
et al. 2012b). de Ruiter and Rudolph (1997) reported three
widely utilized polysaccharides produced by seaweeds: algi-
nates, agars, and carrageenans. Alginates are obtained from
brown seaweed extracts (Ackoo et al. 2001), while agar and
carrageenan are derived from red seaweed extracts (Aguilan
et al. 2003, 2006). The high carrageenan and agar contents of
red seaweeds are what make these organisms so attractive as
sources of bioethanol. Gelidium amansii has also been consid-
ered as a candidate for the production of bioethanol because of
its high sugar content (Jeong and Park 2010; Meinita et al.
2013). Bioethanol production from seaweed requires three

main steps: hydrolysis of polysaccharides into monosac-
charides, fermentation of the monosaccharides to create
bioethanol, and recovery of the bioethanol (Meinita et al.
2012a, b). Chemicals (acid or alkali) and enzymes are typically
applied to break down seaweed polysaccharides. Chemical
(acid) hydrolysis is favorable due to its low cost and high reaction
rate. However, it is more likely to result in undesired by-products
that may affect cell growth and fermentation (Herrera et al. 2004;
Meinita et al. 2012b, c; Wyman 1994).
In the current study, 20 species of seaweed were evaluated
as resources for ethanol production. Additional method re-
finement was performed with Palmaria palmata, the most
promising candidate. The various parameters of acid hydro-
lysis (catalyst amount, hydrolysis temperature, and time) and
fermentation (inoculum concentration, fermentation period,
and medium pH) were then adjusted to obtain the highest
possible ethanol yield from P. palmata.
P. palmata, a red alga, lives in cold and warm temperate
waters in subtidal zones to a depth of up to 20 m (Garbary
et al. 2012). It is commonly referred to as dulse and is
abundant in the North Atlantic and Arctic oceans (Critchley
and Ohno 1998; Guiry and Guiry 2011). It is used as a food
source due to its high carbohydrate content (Rogers 2011)
and has been used as a medicinal supplement for patients
with hyperkalemia because of its high potassium content
(McGrath et al. 2010) and as a free-radical scavenger due
to its high antioxidant level (Li et al. 2011).
Materials and methods
Preparation of seaweed sample Twenty species of seaweed
were collected from Sayang Heulang Beach, West Java, Java
Island, Indonesia. Once collected, the seaweed was washed
with distilled water to remove dirt and salt. The seaweeds
were identified and dried in the sun for 3 days. Dried seaweeds
were ground into powder using a mortar for further study.
Acid hydrolysis Sulfuric acid (H2SO4) was used as the cata-
lyst in acid hydrolysis. Three grams of dried seaweed powder
was combined with 30 mL of 0.2 M H2SO4 in a 250-mL
flask in an autoclave at 121 C for 15 min. After hydrolysis,
residues were separated by centrifugation at 3,000 rpm for
25 min. The upper brown liquid (hydrolysate) was then
removed for analyses of sugars and by-products.
The hydrolysis reaction was optimized by adjusting H2SO4
concentrations (01.0 M), hydrolysis time (030 min), and
hydrolysis temperature (105130 C). After hydrolysis was
complete, the residues were separated as described above. The
sugar content in the liquid was then analyzed.
Fermentation of the hydrolysate The fermentation process
used commercial brewers yeast (freeze-dried Saccharomyces
J Appl Phycol
cerevisiae, Jenico, South Korea) to produce ethanol. The basal
medium consisted of 0.02 % (NH4)2SO4 and 0.006 %
NaH2PO4, adjusted to pH 5 (Prescott and Dun 1959). The
fermentation broth consisted of the hydrolysate and basal
medium at a ratio of 1:2. The fermentation was conducted with
3 mL of broth in an 8-mL bottle for 72 h in a shaking incubator
at 30 C with gentle shaking at 130 rpm. Samples for measur-
ing sugar content and ethanol concentration were obtained at
set times during fermentation.
The fermentation process was optimized by adjusting the
fermentation time (24168 h), inoculum concentration (0.2
2.0 mg mL1), and medium pH (46). Sugar and ethanol
concentrations in the broth were determined using high-
performance liquid chromatography (HPLC) and gas chro-
matography (GC), respectively.
Analysis of sugars, by-products, and ethanol The total car-
bohydrate content of the dried seaweed samples was deter-
mined by a phenolH2SO4 method using carrageenan as a
standard (Kochert 1978). Moisture content was determined by
weighing seaweed samples before and after drying at 105 C
for 18 h until they reached a stable weight. Levels of reducing
sugars were determined using the dinitrosalicylic acid method
(Chaplin 1986). Levels of monosaccharides (galactose and
glucose) and by-products (5-hydroxymethylfurfural (HMF)
and levulinic acid) were measured after hydrolysis using a
HPLC system equipped with an Alltech IOA 1000 column for
organic acids (7.8 mm300 cm). Elution was performed at
60 C using a mobile phase of 0.005 N H2SO4 at a flow rate
0.3 mL min1. Ethanol content was measured after fermenta-
tion by GC (Agilent 6890N, USA) on a 2B-WAX column
(Agilent Technologies, USA). The oven was set to a minimum
temperature of 35 C and a maximum temperature of 200 C
(Meinita et al. 2012a, b).
Analysis of carrageenan content Five gram of dried P.
palmata was washed with tap water to remove sand and salt
and then incubated in 200 mL of 6 % KOH in water at 80 C
for 3 h. The samples were then stirred lightly in 100 mL of
distilled water and boiled for at least 1 h until the sample
disintegrated. The residue was then separated from the filtrate
by centrifugation at 3,000 rpm for 25 min.
Fifty milliliter of 10 % NaCl solution was added to the
filtrate. The filtrate solution was then diluted to double its
volume with ethanol. The resulting carrageenan precipitate
was then soaked in alcohol, dried at 60 C in an oven, and
weighed (Istini et al. 1994).
Results
Table 1 shows the total carbohydrate content, the levels of
sugars and by-products from acid hydrolysis, and a
J Appl Phycol

Table 1 Comparisons of carbohydrates, moisture, reducing sugars, galactose, 5-HMF, levulinic acid, and ethanol yield obtained from the acid
hydrolysate of 20 species of seaweed collected from Sayang Heulang Beach, Indonesia

Seaweed species Relative% in dry tissue Amount (mg g1 seaweed) in hydrolysate Ethanol yield
(mg g1 seaweed)
Carbohydrate Moisture Reducing sugar Galactose 5-HMF Levulinic acid from hydrolysate
(mg g1 seaweed) (% w/w)

Gracilaria coronopifolia 225.95.80 11.20.13 72.21.17 32.91.63 12.50.00 0.50.00 1.00.02

Gelidiella acerrosa 450.64.57 9.50.28 72.50.84 33.30.28 11.10.22 0.70.00 1.20.20
Amphiroa fragilissima 264.129.86 12.60.23 72.51.22 44.02.49 8.30.74 0.40.00 9.21.50
Gigartina affinis 382.510.78 13.70.37 52.00.20 2.80.13 0.20.00 0.40.00 2.00.02
Acanthophora spicifera 445.811.71 12.10.37 72.32.66 12.80.36 5.70.59 0.40.00 1.60.20
Acanthophora muscoides 232.048.27 9.60.17 72.01.55 23.50.13 5.00.59 1.30.10 2.30.30
Palmaria palmata 469.99.36 13.30.54 164.33.17 70.63.40 0.80.44 0.40.00 12.90.20
Galaxaura subfruticulosa 263.04.22 9.60.26 32.81.47 5.40.73 0.20.00 1.00.53 2.10.05
Gracilaria arcuata 469.78.26 8.60.53 71.30.82 23.20.24 7.50.00 0.50.09 2.40.05
Ulfa fasciata 456.34.71 14.60.51 18.21.17 1.50.14 0.20.00 0.40.00 1.90.02
Bornetella nitida 112.615.00 10.20.46 71.01.41 15.90.37 0.20.00 0.40.00 1.90.03
Ulva reticulata 468.15.48 12.50.23 71.21.17 22.70.72 0.50.22 0.40.00 3.10.09
Chaetomorpha antenina 425.211.52 10.40.42 71.31.75 3.51.11 0.40.22 0.40.00 0.80.00
Valoniopsis pachynema 451.49.66 14.60.35 72.01.67 13.41.04 0.20.00 0.40.00 2.10.20
Chaetomorpha crassa 353.67.27 12.80.25 70.21.33 8.90.6 0.20.00 0.40.00 0.10.30
Turbinaria conoides 106.35.03 9.60.45 70.21.17 6.10.96 0.20.00 0.40.00 1.40.06
Sargassum polycystum 119.56.63 11.40.41 70.51.38 5.30.14 0.20.00 0.40.00 1.90.02
Sargassum duplicatum 140.71.97 13.40.51 70.31.51 6.30.27 0.20.00 0.40.00 1.60.10
Padina australis 145.021.45 10.50.40 60.21.60 3.30.48 0.20.00 0.40.00 2.00.09
Dictyopteris sp. 369.73.33 11.60.58 70.00.89 7.70.37 0.20.00 0.40.00 1.10.20

All values were calculated against the dry weight of tissues after removal moisture content. Seaweed powders (10 %) were hydrolyzed with 0.2 M
H2SO4 in an autoclave at 121 C for 15 min. Values represent the mean SD (n3)

comparison of the ethanol obtained after fermentation for
each of the species evaluated. P. palmata showed the
highest potential for ethanol production with the highest
total carbohydrate content and the highest levels of re-
ducing sugars, galactose, and glucose obtained after hy-
drolysis. In contrast, by-product levels (5-HMF and levulinic
acid) in the hydrolysate were the lowest among the 20 sea-
weeds (Table 1). P. palmata also yielded the most ethanol
following fermentation. The total carbohydrate content of P.
palmata was approximately 469 mg g1 seaweed with a
carageenan content of approximately 354 mg g1 seaweed.
Following acid hydrolysis, reducing sugar and galactose
levels in the hydrolysate were 164.3 and 70.6 mg g1 seaweed,
respectively. These high sugar concentrations resulted in a
relatively high ethanol yield (12.9 mg ethanol g1 seaweed).
Thus, P. palmata was chosen for further study and process
optimization.
The results of varying the H2SO4 concentration in the
hydrolysis reaction mixture from 0 to 1 M are shown in
Fig. 1. Each reaction proceeded at 121 C for 15 min. The
reaction containing 0.4 M H2SO4 yielded the highest levels
of reducing sugars (216.1 mg g 1 seaweed), glucose
(32.2 mg g 1 seaweed), and galactose (134.8 mg g 1
seaweed).
Figure 2 shows the results of varying hydrolysis time on
the levels of carbohydrates extracted from P. palmata. In all
cases, hydrolysis was performed in 0.4 M H2SO4 at 121 C
for 030 min. The highest concentrations of reducing sugars
(218.2 mg g1 seaweed), glucose (31.5 mg g1 seaweed), and
galactose (131.2 mg g1 seaweed) were obtained after 25 min
of hydrolysis.
Figure 3 shows that the optimal temperature for acid hy-
drolysis was 125 C. Under these conditions, 218.4 mg g1
seaweed of reducing sugars, 127.6 mg g1 seaweed of galac-
tose, and 27.0 mg g1 seaweed of glucose were obtained in the
hydrolysate of P. palmata.
Fermentation time (24168 h), inoculum concentration
(0.22.0 mg mL1), and medium pH (46) were also varied
and optimized for ethanol production at 30 C in a shaking
incubator at 130 rpm for 72 h. In each experiment, the
fermentation broth was prepared from the hydrolysate of P.
palmata hydrolyzed in 0.4 M H2SO4 at 125 C for 25 min.
Figure 4 shows that the optimal fermentation time was 72 h,
resulting in an ethanol yield of 17.3 mg g1 of seaweed.
 J Appl Phycol

250 250

200 200
Carbohydrates (mg g-1 seaweed)

Carbohydrate (mg g-1 seaweed)

150 150

100 100

50 50

0 0
0,0 0,2 0,4 0,6 0,8 1,0 105 110 115 120 125 130

H2SO4 concentration (M) Temperature (0C)

Fig. 3 The effects of pressure on the amounts of reducing sugars (black

Fig. 1 The effects of H2SO4 concentration on the amounts of reducing
circles), galactose (white circles), and glucose (black triangles) generated
sugars (black circles), galactose (white circles), and glucose (black trian-
during the acid hydrolysis of P. palmata were measured. The hydrolysis
gles) generated during the acid hydrolysis of P. palmata were measured.
was performed in 0.4 H2SO4 in an autoclave at 121 C for 25 min. Each
The hydrolysis was conducted in an autoclave at 121 C for 15 min. Each
value represents the mean of three independent measurements
value represents the mean of three independent measurements

production by fermentation (Table 1). Bioethanol production

Similarly, Fig. 5 shows that the optimal concentration of yeast
for ethanol fermentation was 1.5 mg mL1, resulting in the
production of 15.5 mg ethanol g1 seaweed.
The effects of medium pH were also evaluated, with an
optimum identified at pH 5.25 as shown in Fig. 6. This
resulted in an ethanol yield of 15.7 mg g1 seaweed.
Discussion
Comparisons of total carbohydrates, sugar content, by-products
generated during hydrolysis, and ethanol yield for 20 varieties
of seaweed show that P. palmata is the best suited for ethanol
250
from seaweed consists of hydrolysis of polysaccharides, fer-
mentation, and the recovery of ethanol (Meinita et al. 2012b).
Two methods are commonly used for hydrolysis: acidic and
enzymatic (Meinita et al. 2012b; Wackett 2011). The acids for
hydrolysis are H2SO4, HCl, H3PO4, and C4H4O4 (Dawei et al.
2011; Meinita et al. 2012b), while the enzymes used differ
according to the particular seaweed being processed. For ex-
ample, the hydrolysis of cellulose requires cellulase and -
glucosidase (Wu et al. 2011; Kang et al. 2012). The hydrolysis
of seaweeds containing laminaran, carrageenan, agar, and algi-
nates needs different, specific enzymes such as laminaranase
and agarase (Jang et al. 2012; Yanagisawa et al. 2011; Yun et al.
18

16

200
Ethanol yield (mg g-1 seaweed)

14
Carbohydrates (mg g-1 seaweed)

12

150
10

8
100
6

4
50

0 0
0 5 10 15 20 25 30 40 60 80 100 120 140 160

Hydrolysis time (min) Fermentation time (h)

Fig. 2 The effects of hydrolysis time on the amounts of reducing sugars
(black circles), galactose (white circles), and glucose (black triangles)
generated during the acid hydrolysis of P. palmata were measured. The
hydrolysis was performed in 0.4 H2SO4 in an autoclave at 121 C. Each
value represents the mean of three independent measurements
Fig. 4 The effects of fermentation time on the ethanol yielded from the
acid hydrolysate of P. palmata were measured. The fermentation was
conducted using brewers yeast in a broth of hydrolysate and basal
medium (1:2) at 30 C in a shaking incubator. Each value represents the
mean of three independent measurements
J Appl Phycol

18
of Kappaphycus alvarezii using H2SO4 for ethanol fermenta-
16
tion, with a 10 % (w/v) substrate concentration and 0.2 M
H2SO4 at 130 C for 15 min, the hydrolysate contained
Ethanol yield (mg g-1 seaweed)

14

12
10
8 14.7 g L1 glucose and galactose, respectively, in 3.0 % H2SO4
6
4
2 production consisted of a higher reaction temperature and
0
0,2 0,4 0,6 0,8 1,0 1,2 1,4
Concentration of brewer's yeast (mg mL )
Fig. 5 The effects of brewers yeast concentration on the ethanol
yielded from the acid hydrolysate of P. palmata were measured. The
fermentation was conducted using brewers yeast in a broth of hydro-
lysate and basal medium (1:2) at 30 C in a shaking incubator. Each
value represents the mean of three independent measurements
2011). Despite the high selectivity of enzymes for seaweed
hydrolysis, acid hydrolysis is generally favored due to its low
cost and high hydrolysis rate (Jeong and Park 2010; Meinita
et al. 2012b, c). However, acid hydrolysis is relatively complex
because the substrate is a solid while the catalyst is in the liquid
phase. The sugar productivity of acid hydrolysis is affected by
several factors including the substrate ratio, type and concen-
tration of acid, and hydrolysis temperature and time (Meinita
et al. 2012b). In the current study, 218.4 mg g1 seaweed of
reducing sugars, 127.6 mg g1 seaweed of galactose, and
27 mg g1 seaweed of glucose were obtained by the acid
hydrolysis of P. palmata in 0.4 M H2SO4 at 125 C for
25 min. Meinita et al. (2012b) reported that in the hydrolysis
18
16
14
Ethanol yield (mg g-1 seaweed)
38.4 mg L1 of reducing sugars, 23.9 g L1 of galactose, and
0.9 g L1 of glucose. Jeong and Park (2010) reported that their
hydrolysis of the marine alga G. amansii produced 2.9 and
at 139.4 C for 15 min. The same process in 3.0 % H2SO4 at
108.2 C for 45 min produced 16.1 g L1 galactose and
2.4 g L1 glucose. Thus, the optimized conditions for glucose
shorter reaction time than those for galactose production.
1,6 1,8 2,0 Producing bioethanol from seaweed hydrolysate requires
-1 fermentation by ethanogenic bacteria or yeast. The bacteria
commonly employed are Escherichia coli (Rude and
Schirmer 2009) and Zymomonas mobilis (Fu et al. 2009).
The most common yeasts employed for ethanol production
are S. cerevisiae (Hanshem and Darwish 2010; Matsushika
and Sawayana 2011; Prasetyo et al. 2011) and Pichia stipitis
(Fu et al. 2009). In this work, commercial S. cerevisiae was
used because galactose is a major constituent of P. palmata
hydrolysate. The fermentation of the acid hydrolysate of P.
palmata resulted in 15.7 mg g1 seaweed of ethanol with an
inoculum concentration of 1.5 mg mL1 at 30 C and 72 h in
a shaking incubator at 130 rpm. Jang et al. (2012) reported a
69.1 % total saccharification yield in the ethanol fermenta-
tion of the brown seaweed Saccharina japonica (10 % [w/v]
seaweed slurry, 40 mM H2SO4, and 1 g dry cell weight
[dcw]L1 Bacillus sp. JS-1). In addition, an ethanol concen-
tration of 7.7 g L1 (theoretical yield of 33.3 %) was obtained
by simultaneous saccharification and fermentation with
0.39 g dcw L1 Bacillus sp. JS-1 and 0.45 g dcw L1 of the
yeast Pichia angophorae KCTC 17574.
The presence of fermentation-inhibiting compounds in the
hydrolysate can hamper the fermentation process (Meinita
et al. 2012c; Qureshi et al. 2008). According to Meinita
et al. (2012c), such compounds include 5-HMF and levulinic

acid. These compounds are generated during acid hydrolysis

12
at high temperatures and may negatively impact cell growth
10 and ethanol production (Herrera et al. 2004; Meinita et al.
8
2012c; Wyman 1994). The inhibitory effects of these com-
pounds can be minimized by adding activated charcoal and
6
calcium hydroxide to the fermenting mixture (Meinita et al.
4 2012c; Mussatto and Roberto 2001). This detoxification pro-
2
cess can increase ethanol concentrations and the overall pro-
ductivity of a given seaweed hydrolysate.
0
4,0 4,5 5,0 5,5 6,0
The current study evaluated the factors that influence the
pH for fermentation
acid hydrolysis and fermentation of P. palmata. Carrageenan,
which is composed of galactose repeat units, was easily hy-
Fig. 6 The effects of pH on the ethanol yielded from the acid hydro- drolyzed and the products fermented to ethanol by yeast.
lysate of P. palmata were measured. The fermentation was conducted
using 1.5 mg mL1 of brewers yeast in a broth of hydrolysate and basal
Thus, the carrageenophyte P. palmata is a promising candi-
medium (1:2) at 30 C in a shaking incubator. Each value represents the date resource for bioethanol production after optimization of
mean of three independent measurements the hydrolysis (saccharification) and fermentation processes.

In conclusion, of the 20 species of seaweed evaluated, P.
palmata showed the highest potential for the large-scale
production of bioethanol. Acid hydrolysis in 0.4 M H2SO4
at 125 C for 25 min produced the highest carbohydrate
(reducing sugars, galactose, and glucose) yield. Fermentation
of this hydrolysate for 72 h with a 1.5 mg mL1 inoculation
concentration of commercial brewers yeast (freeze-dried S.
cerevisiae) at pH 5.25 produced the highest yield of ethanol.
Acknowledgments We thank the Marine Biotechnology Laboratory
& Biochemical Engineering Laboratory of Pukyong National Univer-
sity and Research Laboratory of Jenderal Soedirman University for the
use of their equipment and research support in the scheme of Interna-
tional Collaboration Research. We also thank the Indonesian Ministry
of National Education (DIKTI) for providing the funding for this study
through its scholarship, Beasiswa Unggulan for graduate support (SM).
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