J Appl Phycol (2018) 30:143–147

https://doi.org/10.1007/s10811-017-1297-x

9TH ASIA PACIFIC CONFERENCE ON ALGAL BIOTECHNOLOGY - BANGKOK

Comparison of bioethanol production from cultivated

versus wild Gracilaria verrucosa and Gracilaria gigas
Maria Dyah Nur Meinita 1 & Bintang Marhaeni 1 & Diyah Fatimah Oktaviani 1,2 &
Gwi-Taek Jeong 3 & Yong-Ki Hong 3

Received: 31 December 2016 / Revised and accepted: 1 October 2017 / Published online: 23 October 2017
# Springer Science+Business Media B.V. 2017

Abstract The seaweed genus Gracilaria is a potential can- wild G. gigas is a promising challenge for aquaculture to avoid
didate for the production of bioethanol due to its high car- overexploitation of this wild seaweed resource.
bohydrate content. Gracilaria is abundant throughout the
world and can be found in both wild and cultivated forms. Keywords Seaweed . Bioethanol . Wild . Cultivated .
Differences in the ecological factors such as temperature, Gracilaria . Rhodophyta
salinity, and light intensity affecting wild and cultivated
specimens may influence the biochemical content of sea-
weeds, including the carbohydrate content. This study Introduction
aimed to investigate the proximate composition and poten-
tial bioethanol production of wild and cultivated G. gigas Agarophyte seaweeds belonging to the red algal genus
and G. verrucosa. Bioethanol was produced using separate Gracilaria are important raw materials in agar production.
hydrolysis fermentation (SHF), employing a combination of Since the beginning of the twentieth century, agar production
enzymatic and acid hydrolysis, followed by fermentation with has relied largely on Gelidium, as it produces a higher quality
Saccharomyces cerevisiae ATCC 200062. The highest carbo- agar (less sulfated) than Gracilaria (Armisen 1995). The
hydrate content was found in wild G. gigas. The highest galac- resulting high demand for Gelidium eventually exceeded the
tose and glucose contents (20.21 ± 0.32 and 9.70 ± 0.49 g L−1, supply of naturally occurring Gelidium, and cultivation of the
respectively), as well as the highest production of bioethanol genus proved problematic. On the other hand, Gracilaria is
(3.56 ± 0.02 g L−1), were also found in wild G. gigas. Thus, we successfully being cultivated in land-based ponds in several
conclude that wild G. gigas is the most promising candidate for countries; the largest producer of which is Indonesia, followed
bioethanol production. Further research is needed to optimize by China and Chile (McHugh 2003; Pambudi et al. 2010;
bioethanol production from wild G. gigas. Domestication of Bixler and Porse 2011, Porse and Rudolph 2017).
Furthermore, Gracilaria cultivation has been steadily increas-
ing, from 936,000 t in 2005 to 3,752,000 t in 2014 (McHugh
2003; Pambudi et al. 2010; Bixler and Porse 2011). Also, the
* Maria Dyah Nur Meinita
maria.meinita@unsoed.ac.id quality of agar produced from Gracilaria can be improved by
pretreating the seaweed with alkali. The successful combina-
tion of Gracilaria cultivation and alkali pretreatment has led
1
Fisheries and Marine Science Faculty, Jenderal Soedirman to Gracilaria replacing Gelidium as the leading source for
University, Jl. Dr. Suparno, Karangwangkal, Purwokerto 53122,
Indonesia
agar production (Armisen 1995).
2
The most common species of Gracilaria used for commer-
Graduate School of World Fisheries University Programme,
Pukyong National University, Namgu, Busan 48513, Republic of
cial agar production are G. verrucosa and G. gigas, which are
Korea obtained from both cultivation and the wild. Recently,
3
Department of Biotechnology, College of Fisheries Science,
Gracilaria species have been used as raw material for
Pukyong National University, Namgu, Busan 48513, Republic of bioethanol production (Kumar et al. 2013; Meinita et al. 2013;
Korea Wu et al. 2014). The cell walls of Gracilaria consist of cellulose
144 J Appl Phycol (2018) 30:143–147

and complex agar polysaccharide; these, in turn, comprise a Microorganisms and medium
mixture of neutral polymer agarose, pyruvated agarose, and sul-
fated galactans, which can be hydrolyzed to sugars and Prior to fermentation, the hydrolysate samples were adjusted
fermented to ethanol. The purpose of the present study was to to pH 5 using 10 N NaOH. Saccharomyces cerevisiae ATCC
compare the biochemical compositions of wild and cultivated 200062 was used for the fermentation. The inoculum culture
G. verrucosa and G. gigas as well as their respective potentials was prepared in a medium containing 10 g yeast extract, 6.4 g
as raw materials for bioethanol production. urea, and 20 g glucose per liter (Meinita et al. 2017). The
resulting yeast suspension was incubated at 30 °C in a shaking
incubator at 130 rpm. Fermentation was carried out in tripli-
Material and methods cate in 100 mL Erlenmeyer flasks, each with a working vol-
ume of 50 mL. The flasks were periodically sampled for mea-
Preparation of seaweed samples surements of sugar and ethanol content.

Cultivated samples of Gracilaria. gigas and G. verrucosa Sugar and ethanol determination
were obtained from Brebes, Central Java, Indonesia, and wild
samples of the same were obtained from Garut Beach, West Monosaccharide composition (galactose, glucose) was deter-
Java, Indonesia. The samples were washed with water and mined by HPLC using an Alltech IOA 1000 organic acid
then freeze-dried. column (300 × 7.8 mm) equipped with an RI detector and
maintained at 60 °C. Ethanol production was measured using
Proximate analysis an Agilent model 6890N Series Gas Chromatograph with a
2B-WAX column (Agilent, USA). The injection volume was
The proximate compositions of cultivated and wild G. gigas 2 μL with an inlet split ratio of 30:1. The initial and maximum
and G. verrucosa were analyzed as follows. The total carbo- oven temperatures were 35 and 250 °C, respectively (Meinita
hydrate expressed as agar was determined by the phenol- et al. 2013).
sulfuric acid method (Kochert 1978). Total lipid was extracted
using a mixture of hexane and isopropanol (3:2) and was
quantified gravimetrically (Radin 1981). The amount of solu- Results and discussion
ble protein in the tissue was estimated according to the method
of Lowry et al. (1951) after heating a tissue suspension at Proximate composition
100 °C in 1 N NaOH for 2 h to obtain complete solubilization
of the protein. Bovine serum albumin was used as a protein The proximate compositions (carbohydrate, protein, lipid, and
standard. The ash content was determined by weighing the ash contents) of cultivated versus wild G. gigas and
residue after heating the sample for 5 h at 575 °C. G. verrucosa are shown in Table 1. In both the cultivated
and wild samples, carbohydrate and ash were the most abun-
Pretreatment dant components, whereas lipid was the least abundant com-
ponent. These results agree with those of previous studies on
H2SO4 was used to hydrolyze carbohydrates into simple the biochemical compositions of seaweeds (Matanjun et al.
sugars. Samples consisting of 10 g powdered waste agar in 2009; Norziah and Ching 2000; Wong and Cheung 2000;
100 mL of 0.2 M H2SO4 in a 250 mL flask were autoclaved at Marinho-Soriano et al. 2006; Gómez-Ordóñez et al. 2010;
120 °C for 15 min (Meinita et al. 2013). The acid-hydrolyzed Vergara-Rodarte et al. 2010; Syad et al. 2013).
slurries of agar wastes were then analyzed for sugar content
using high-performance liquid chromatography (HPLC) and Carbohydrate
used in the next steps.
Carbohydrate was the major component in the proximate
Enzymatic saccharification composition of all the Gracilaria seaweeds examined. The
carbohydrate content ranged from 38.38 ± 7.77% in cultivated
The acid-hydrolyzed slurries from the previous step were ad- G. verrucosa to 64.71 ± 3.74% in wild G. gigas. The highest
justed to pH 5 and then saccharified using an enzyme mixture observed carbohydrate concentration was comparatively
of Cellic C tec II, Viscozyme, and Cellic H tec II (Meinita et al. greater than that reported by Norziah and Ching (2000) for
2017). The saccharification was carried out at 50 °C in a water G. changgi, but it was similar to that obtained by Marinho-
bath shaker at 130 rpm for 24 h. The resulting hydrolysates Soriano et al. (2006) for G. cervicornis (63.13 ± 3.50%). For
were then evaluated for sugar content using HPLC and used in both G. verrucosa and G. gigas, the carbohydrate content was
the next steps. greater in the wild samples than in the cultivated samples. The
J Appl Phycol (2018) 30:143–147 145

Table 1 Proximate analysis (%)

of wild and cultivated Gracilaria Component Gracilaria verrucosa Gracilaria gigas
verrucosa and G. gigas. Values
represent the mean ± SD (n ≥ 3) Wild Cultivated Wild Cultivated

Carbohydrate 60.81 ± 3.91 38.38 ± 7.77 64.71 ± 3.74 47.31 ± 9.20

Protein 9.86 ± 3.79 6.64 ± 3.21 12.63 ± 1.58 8.14 ± 2.31
Lipid 0.80 ± 0.09 0.58 ± 0.10 1.31 ± 1.25 0.60 ± 0.07
Ash 13.85 ± 0.99 12.51 ± 0.15 19.59 ± 0.15 17.86 ± 0.98

range of carbohydrate values was comparable to that seen in In this study, the cultivated and wild G. gigas and
previous studies of the carbohydrate content of red seaweeds G. verrucosa differed in protein, carbohydrate, lipid, and ash
(Norziah and Ching 2000; Wong and Cheung 2000; Marinho- contents. These differences might be attributed to environ-
Soriano et al. 2006; Matanjun et al. 2009; Gómez-Ordóñez mental parameters. Some researchers have concluded that
et al. 2010; Vergara-Rodarte et al. 2010; Syad et al. 2013). the proximate compositions of seaweeds vary with different
species and seasons (Marinho-Soriano et al. 2006; Ratana-
arporn and Chirapart 2006; Matanjun et al. 2009; Denis
Protein
et al. 2010).
The protein content of the Gracilaria seaweeds ranged from
6.64 ± 3.21 to 12.63 ± 1.58%, comparable to the values re- Sugar content before and after enzymatic hydrolysis
ported by Norziah and Ching (2000) for G. changgi (6.9%)
but lower than those reported by Marinho-Soriano et al. We increased the content of monosaccharides in our sea-
(2006) for G. cervicornis (22.96%). The protein range report- weed samples by two methods, acid hydrolysis followed
ed here is in agreement with that described in most Gracilaria by enzymatic saccharification, to convert polysaccharides
studies. According to Briggs and Smith (1993), the protein into simple sugars. Previous studies (Meinita et al. 2013,
content in most Gracilaria is between 7 and 13%. 2015) showed that dilute acid hydrolysis using H2SO4 is
the most effective method for hydrolyzing the polysaccha-
rides of red seaweed. Enzymatic saccharification is an im-
Lipid
portant step in bioethanol production, with many advan-
tages over chemical saccharification. In enzymatic sacchar-
The lipid content of the Gracilaria seaweeds ranged from
ification, mild pH and temperature are applied. These mild
0.58 ± 0.10 to 1.31 ± 1.25%.
conditions avoid producing the toxic and/or corrosive by-
The maximum lipid content was observed in wild G. gigas,
products that may result from acid hydrolysis (Alvira et al.
followed by wild G. verrucosa, cultivated G. gigas, and cul-
2010). The sugar contents before and after enzymatic sac-
tivated G. verrucosa, in that order. Generally, seaweeds are
charification of wild G. verrucosa, cultivated
relatively low in lipid. Previous studies of the lipid content of
G. verrucosa, wild G. gigas, and cultivated G. gigas are
Gracilaria species reported values ranging from 0.028 to
shown in Fig. 1. The galactose content of the wild
3.3% (Norziah and Ching 2000; Marinho-Soriano et al.
G. verrucosa, cultivated G. verrucosa, wild G. gigas, and
2006; Syad et al. 2013). Compared to other biochemical com-
cultivated G. gigas samples after acid hydrolysis were
ponents of seaweeds, lipid appears to be the least abundant.
15.76 ± 0.18, 10.26 ± 0.16, 18.54 ± 18.54, and
Moreover, the lipid content of tropical seaweeds is generally
14.08 ± 0.16 g L−1, respectively. After enzymatic sacchar-
lower than that of subtropical seaweeds.
ification, the galactose content of the wild G. verrucosa,
cultivated G. verrucosa, wild G. gigas, and cultivated
Ash G. gigas samples were 16.84 ± 0.24, 12.49 ± 0.09,
20.21 ± 0.32, and 15.20 ± 0.16 g L−1, respectively. The
The ash contents of wild G. gigas, cultivated G. gigas, wild glucose content of the wild G. verrucosa, cultivated
G. verrucosa, and cultivated G. verrucosa were G. verrucosa, wild G. gigas, and cultivated G. gigas sam-
19.59 ± 0.15, 17.86 ± 0.98, 13.85 ± 0.99, and ples after acid hydrolysis were 0.66 ± 0.02, 0.40 ± 0.03,
12.51 ± 0.15%, respectively. These values are within the 1.45 ± 0.03, and 1.01 ± 0.05 g L−1, respectively. After
range reported for most Gracilaria seaweeds by other re- enzymatic saccharification, the glucose content of the wild
searchers (Norziah and Ching 2000; Marinho-Soriano et al. G. verrucosa, cultivated G. verrucosa, wild G. gigas, and
2006; Syad et al. 2013). In general, ash content represents cultivated G. gigas samples were 6.47 ± 0.25, 2.67 ± 0.28,
the total mineral content of the seaweeds. 9.70 ± 0.49, and 4.37 ± 0.16 g L−1, respectively. Based on
146 J Appl Phycol (2018) 30:143–147

5
a Galactose (g L-1)
25
Galactose before enzymatic saccharification 20
Glucose (g L-1)
Galactose after enzymatic saccharification
EtOH (g L-1) 4

Glucose and galactose (g L-1)

20
15

EtOH (g L-1)
3
Galactose (g L )
-1

15

10
2
10

5 1
5

0 0 0
Gv wild Gv cult Gg wild Gg cult 0 3 6 9 12 24 48 72 96

b Time (h)

12
Fig. 2 Time course of ethanol production and galactose and glucose
Glucose before enzymatic saccharification
Glucose after enzymatic saccharification utilization during fermentation of an enzymatic hydrolysate of G. gigas.
10
Values represent the mean ± SD (n ≥ 3)

bioethanol production (3.56 g L−1) after 24 h of fermenta-

Glucose (g L )

8
-1

tion, which corresponded to a 28.81% theoretical yield.

6
Gracilaria species may be the best agarophytes to use
4 for bioethanol production due to their abundant carbohy-
drate content. Among cultivated and wild G. gigas and
2 G. verrucosa, wild G. gigas appears to be the most prom-
ising species, based on proximate composition and sugar
0
Gv wild Gv cult Gg wild Gg cult content after enzymatic saccharification. Carbohydrate is
Fig. 1 Galactose (a) and glucose (b) content of wild Gracilaria the most abundant component found in wild G. gigas.
verrucosa (Gv wild), cultivated G. verrucosa (Gv cult), wild G. gigas Acid hydrolysis, followed by enzymatic saccharification,
(Gg wild), and cultivated G. gigas (Gg cult) before and after enzymatic
significantly increased the sugar content of the wild
saccharification. Values represent the mean ± SD (n ≥ 3)
G. gigas samples. Currently, most of the G. gigas supply
is from the wild. Hence, domestication of G. gigas is a
these results, we conclude that enzymatic saccharification promising approach to aquaculture designed to facilitate
is the more efficient method for increasing glucose, rather pilot-scale production of bioethanol. Domestication of wild
than galactose, in Gracilaria seaweeds. The enzymatic hy- G. gigas may be needed to avoid collapse of the wild sea-
drolysate of wild G. gigas contained the greatest concen- weed population due to overexploitation.
tration of glucose; suggesting wild G. gigas is the best
candidate for bioethanol production; hence, it was used
for the subsequent ethanol fermentation.
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