Biomass and Bioenergy 90 (2016) 148e154

Contents lists available at ScienceDirect

Biomass and Bioenergy

journal homepage: http://www.elsevier.com/locate/biombioe

Research paper

Characterization, pretreatment and saccharication of spent seaweed

biomass for bioethanol production using baker's yeast
M.P. Sudhakar a, Ravel Merlyn a, K. Arunkumar b, K. Perumal a, *
a

Shri A.M.M. Murugappa Chettiar Research Centre, Taramani, Chennai, 600113, Tamilnadu, India
Marine Algae Research Division, Post Graduate and Research Department of Botany, Alagappa Government Arts and Science College, Alagappa University,
Karaikudi, Tamilnadu, India

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 9 April 2015
Received in revised form
4 February 2016
Accepted 23 March 2016

Seaweeds are marine macroalgae found abundantly and viewed as potential source of phycocolloids to
produce biofuel. In this study, seaweed spent biomass obtained from alginate production industry and
biomass obtained after pigment extraction were found to contain a considerable amount of phycocolloids. These two spent biomasses were investigated for the production of ethanol. In this study, the
red seaweed spent biomass of Gracilaria corticata var corticata showed higher content of polysaccharide
(190.71 30.67 mg g
1 dry weight) than brown seaweed spent biomass (industrial)
(136.28 30.09 mg g
1 dry weight). Hydrolysis of spent biomasses with different concentrations of
sulfuric acid (0.1%, 0.5% and 1%) was also investigated. Brown seaweed spent biomass and red seaweed
spent biomass exhibited high amount of sugar in 0.5% and 1% sulfuric acid treatment, respectively.
Proximate and ultimate composition of seaweed spent biomasses were analysed for energy value. The
FT-Raman spectra exhibited similar stretches for both acid hydrolysed spent biomasses with their
respective standards. Ethanol produced through a fermentation process using spent hydrolysates with
baker's yeast at pH 5.3 was found to be signicant. The ethanol yield from brown seaweed spent biomass
and red seaweed spent biomass was observed to be 0.011 g g
1 and 0.02 0.003 g g
1 respectively, when
compared with YPD (0.42 0.03 g g
1) and D-galactose (0.37 0.04 g g
1) as standard on day 4. The
present study revealed the possibility of effective utilization of spent biomass from seaweed industry for
ethanol production.
2016 Elsevier Ltd. All rights reserved.

Keywords:
Seaweed spent
Gracilaria corticata
Mild-acid treatment
Fermentation
Baker's yeast
Bioethanol

1. Introduction
Fossil fuels accounted for about 88% of the global primary energy consumption [1]. The depletion of fossil fuels, increased cost of
fuels, concern about global climatic changes and increased CO2
emission have led to the discovery of bio-fuels [2]. Biofuels are
liquid or gaseous fuels produced from plants, including microalgae,
and seaweeds [3], municipal wastes [4] and agricultural or forest
by-products [5,6]. Among biofuels, bioethanol, a renewable source
of energy, has been accepted more widely as an alternative to fossil
fuels.
Seaweeds are macroalgae found abundantly on east and west

Abbreviations: BS, brown seaweed spent; RS, red seaweed spent; DW, dry
weight.
* Corresponding author.
E-mail address: perumalk@mcrc.murugappa.org (K. Perumal).
http://dx.doi.org/10.1016/j.biombioe.2016.03.031
0961-9534/ 2016 Elsevier Ltd. All rights reserved.

coast of India and broadly classied into red, brown and green
forms based on colour and biochemical composition [7]. Seaweeds
have many bioactive compounds like pigments, sulfated polysaccharides such as agar, carrageenan and alginates, that are used
for various industrial applications [8,9]. Gracilaria sp., Kappaphycus
sp. and Sargassum sp., are well known for agar, carrageenan and
alginates production at an industrial scale level, respectively.
Nearly 7.5e8 million tonnes of wet seaweeds are harvested
worldwide per year [10]. The production of macroalgae is
15.5 million tonne fresh weight per annum and contributes 93%
commercial value of seaweeds in 2008 worldwide [11e14]. Saccharina latissima (previously known as Laminaria saccharina) is the
fastest-growing seaweed called gigantic kelp species. This seaweed
is similar toSaccharina japonica of which 4 million tonnes fresh
weight is harvested annually in Northern China, and almost
0.3 million tonne fresh weight of S. japonica was also harvested in
Korea whereas from Japan reported for about 50,000 tonnes
[9,15e17]. Commercially important seaweeds such as Gracilaria sp.,

M.P. Sudhakar et al. / Biomass and Bioenergy 90 (2016) 148e154

Sargassum sp., were cultivated long before since 1960 and 1970 in
India and even today Central Salt and Marine Research Institute
(CSMCRI), Mandapam and Bhavnagar, India successfully cultivated
Gracilaria edulis commercially and developed a technology to grow
onshore [10,18,19].
Agar (1179 tonne) and alginate (3180 tonne) production in India
seems to be very less when compared to world scenario up to
2003e2004 [10]. The huge amount of spent biomass generated
from seaweed industry worldwide and effective utilization of those
spent is really a challenging task. In India, some seaweed industries
are converting the spent seaweed biomass to agricultural manures
through composting. Though composting is a simple process, the
current status of biofuel production through waste has become an
innovative method to convert waste into a more valuable product.
The seasonal variations in algal sugar quantity may vary
depending upon climatic conditions. The phycocolloids such as
agar, carrageenan and alginates after acid pretreatment can be
effectively utilised by yeast for ethanol production [20e22]. However, yeast has a narrow substrate range such as six-carbon sugars
[23] for its growth and ethanol production. Mostly seaweed yields
ve-carbon and six-carbon sugars on hydrolysis by acid or alkali. In
order to convert polysaccharides to monosugars, an effective pretreatment process is necessary. Mild acid treatment was found to
be effective on hydrolysis of polysaccharides at a particular temperature [24e26]. Saccharication and hydrolysis are essential for
bioconversion of substrate. Enzymatic hydrolysis is a method for
converting of polysaccharides into monosaccharides and can be
widely used for ethanol production but cost increases invariably
[27]. Acid cleaves b-1-4-glycosidic bond of cellulose and other
complex polysaccharides [28]. Monosugars formed due to hydrolysis are found to be valuable substrates for bioethanol production
[29e31].
Ethanol production of hexose sugars is easy and redeox
balanced, while production from pentoses or mannitol generates
surplus hydrogen. Many bacteria overcome by transhydrogenase
production, but yeast cannot produce transhydrogenase to solve
this problem. Yeasts can overcome the problem by controlling
supply of oxygen, which leads to complete oxidation of the sugar to
CO2 and water, and reduces ethanol yields [32e34].
The ethanol production has been reported from seaweeds such
as green (Ulva lactuca, Ulva pertusa); red (Kappaphycus alvarezii,
Gelidium amansii, Gelidium elegans, Gracilaria salicornia); and
brown (Laminaria japonica, Laminaria hyperborea, Saccharina latissima, Sargassum fulvellum, Undaria pinnatida, Alaria crassifolia)
[35]. Apart from whole seaweeds, the industrial spent, such as
oating residues, spent biomass can also be used for ethanol production [5].
The prospective of ethanol production from seaweeds is based
on the carbohydrate content (60% of dry weight) and a conversion
(90%) ratio to produce ethanol. Through fermentation, 1 g of sugar
can yield 0.4e0.5 g of ethanol or 0.22 kg or 0.27 L ethanol from 1 kg
dry weight seaweed biomass, equivalent to approximately 0.05 L
ethanol per kg wet weight [34].
However, the global demand for bioethanol continues to be an
interesting research for human benet and industrial applications.
World production of bioethanol reached over 51,000 million litres
in 2007 [36]. The world ethanol market is projected to reach
100 billion litres per annum by the year 2012 [37]. Global demand
of ethanol is currently 86 billion litres [38].
The present study is aimed at production of ethanol by utilizing
spent biomass generated from seaweed processing industry using
baker's yeast and its potential of converting galactose and alginate
monomers to bioethanol through fermentation.

149

2. Materials and methods

2.1. Seaweed spent collection and processing
Brown seaweed spent (BS) biomass was collected from SNAP
Alginates and Natural Products Pvt. Ltd., Ranipet, Tamilnadu, India.
The collected spent was dried under sunlight to remove water
content up to 80e90%. The dried spent was powdered using a mixer
grinder and sieved in 100 mesh sizes for further use. Red seaweed
spent (RS) biomass were prepared in the laboratory. Red seaweed
Gracilaria corticata var corticata was collected from a Manapad
coastal area, Tamilnadu, India. The phycobiliproteins were extracted using 0.1 M potassium phosphate buffer [39e41] and the
remaining spent was processed similar to industrial spent (BS) and
utilised for further experiment. The processed spent biomass was
stored at room temperature.

2.2. Proximate, ultimate and biochemical analysis of seaweed spent

biomass
The collected spent biomass was screened for pH, moisture
content, ash content, volatile matter, xed carbon, C, H, N, S analysis
and total organic carbon [42]. Apart from these parameters, the
biochemical content of spent biomass, such as total carbohydrates
[43], reducing sugar [44], total phenol [45] and total protein [46,47],
was quantied as explained below.

2.2.1. Analysis of pH
BS biomass collected from industry contains water along with
biomass and appears to be semi solid in nature which was analysed
using pH meter at room temperature; RS biomass prepared in the
laboratory (after extraction of phycobiliproteins using buffer), obtained as solid biomass, was analysed using pH paper at room
temperature.

2.2.2. Moisture analysis

One gram of spent biomass was taken in a known weight of
crucible and dried in a hot air oven at 60  C till the constant weight
is attained. The moisture content was calculated by using the formula below.

Moisture % Initial biomass weight

Final biomass weight
 =Initial biomass weight  100

2.2.3. Analysis of total carbohydrates

Known quantity (100 mg) of spent biomass was taken into a
boiling tube and hydrolysed by keeping in boiling water bath for 5 h
with 5 mL of 2.5 N HCl and then cooled to room temperature. The
hydrolysate was neutralized with solid sodium carbonate until the
effervescence ceases and the volume was made up to 100 mL, then
centrifuged. The supernatant 0.2 mL was taken in three test tubes,
the volume of each tube was made up to 1 mL with distilled water.
Distilled water was used as blank. About 1 mL of 5% phenol was
added to each tubes including blank followed by the addition of
5 mL 96% sulfuric acid to each tube. The content in the tubes was
shaken well and incubated at 25e30  C for 20 min. The absorbance
was recorded at 490 nm. The amount of total carbohydrate present
in the sample solution was calculated using glucose as standard and
was expressed in mg g
1 [43].

150

M.P. Sudhakar et al. / Biomass and Bioenergy 90 (2016) 148e154

2.2.4. Reducing sugar analysis

About 100 mg of biomass was extracted with 80% hot ethanol
twice (5 mL each time). The supernatant was collected and evaporated in a water bath at 80  C. Ten milliliters of water was added to
dissolve the sugars. About 0.1 mL of sample was pipetted out in test
tubes and the volume was made up to 1 mL using distilled water;
1 mL of distilled water served as blank. DNS reagent (3 mL) was
added to each tube including blank. The tubes were kept in boiling
water bath for 5 min. When the contents in the tubes were still
warm, 1 mL of 40% Rochelle salt was added. Development of dark
red colour indicates the presence of reducing sugar; and the intensity of the colour was read in UVevisible spectrophotometer at
510 nm and compared with the standard. In case of fermented
samples, 2 mL of the fermented broth was collected in eppendorf
tubes. Samples were centrifuged at 10,000 rpm for 20 min. The
supernatant was collected in fresh eppendorf tubes and the
reducing sugar was analysed as mentioned above throughout the
experiment [44].
2.2.5. Total phenol content in spent biomass
About 100 mg of the spent biomass was taken and ground in a
mortar and pestle with 10 mL of distilled water. The sample was
collected and centrifuged at 8000 rpm for 15 min. The supernatant
was collected in a fresh centrifuge tube. About 100 mL of sample
(supernatant) were taken in test tubes and the volume was made to
1 mL with distilled water; 1 mL of distilled water served as blank. To
each tube, 2 mL of 2% Na2CO3 was added including the blank. The
tubes were allowed to stand for 2 min at room temperature; 100 mL
of 50% FolineCiocalteau reagent was added to each tube and the
contents in the tubes were mixed thoroughly. The tubes were
incubated in dark for 30 min. The intensity of the colour was read in
UVeVisible Spectrophotometer at 720 nm against a blank. A series
of standard using Gallic acid was run simultaneously and a graph
was plotted. The amount of total phenol present in the water
extract of the sample was calculated from the standard graph [45].
2.2.6. Total protein content in spent biomass
Spent biomass of 100 mg was taken for this study. The biomass
was ground with mortar and pestle using distilled water. The homogenate was centrifuged at 15,000 rpm for 20 min at 4  C. Supernatant was collected and stored at 4  C. About 2 mL of 0.1 N
NaOH was added to the pellet and was allowed to stand at room
temperature for 1 h under occasional shaking, and then again
centrifuged at 15,000 rpm at room temperature. The obtained supernatant was added to the rst collected supernatant, which is the
crude protein. This crude protein was precipitated using 25% TCA
(TCA: homogenate 2.5:1 v/v) in an ice bath for 30 min followed
by centrifugation at 15, 000 rpm. The supernatant was discarded
and the pellet was extracted twice as above with 10% TCA and 5%
TCA. Each time supernatant was discarded and the protein pellet
obtained was used for total protein analysis using BSA as a standard
[46,47].
2.3. Pretreatment of spent biomass using H2SO4
Mild acid treatment was optimised for hydrolysis of polysaccharides from spent biomass [26,29,48]. About 10 mL of 0.1%,
0.5% and 1% H2SO4 was used to hydrolyse 0.2 g of spent biomass
and autoclaved at 121  C for 15 min. The hydrolysed spent biomasses were ltered using muslin cloth after cooling to room
temperature. The ltrate was then centrifuged at 10,000 rpm for
20 min to remove insoluble substances present in the hydrolysates.
The pH of the ltrate was 1.2e1.5 and it was adjusted to 5.3 using
1 M NaOH for fermentation. The hydrolysates were subjected to FTRaman analysis to conrm the presence of sugars and its converted

form after pretreatment. Further, the quality of hydrolysate was

assessed using FT-Raman spectra. Based on FT-Raman spectra, the
method of hydrolysis can also be optimized. Hydrolysis of different
quantities of seaweed spent biomass with a standard amount of
acid concentration was carried out to optimize the concentration of
biomass to extract the maximum amount of sugar from the hydrolysates. For this, different quantities of BS biomass such as 2, 4, 6,
8 and 10 g were used in 50 mL of 1% H2SO4 and autoclaved at 121  C
for 15 min.
2.4. Fermentation process
Commercially available baker's yeast was used for saccharication. The yeast pellet was revived in Yeast extract Peptone Dextrose
(YPD) broth (Hi-Media) and subsequently sub-cultured for further
use. The colonies were maintained on slants of YPD agar. Supplementary nutrients in g L
1: Urea, 1; Na2HPO4, 0.5; KH2PO4, 2.5;
MgSO4, 1; (NH4)2SO4, 1; FeSO4, 0.001 were added to the hydrolysates before inoculation of 10% v/v of yeast cells [5]. The substrate
(spent biomass) concentration was maintained as 5.6 g in 70 mL of
acid. Hundred milliliter-glass vials containing 70 mL of sterile hydrolysate was inoculated with 7 mL of yeast and incubated at
30e34  C under shaking condition at 50 rpm. The vials were sealed
with rubber cork and crimps. YPD broth (HiMedia) and D-galactose
(SRL) at a concentration of 20 g L
1 were used as standards for
fermentation to compare the production of ethanol with that of
seaweed spent. The temperature was maintained at 30e34  C and
under shaking (50 rpm) for an incubation period of 5 days.
2.5. Analysis of sugar and ethanol
The reducing sugar present in hydrolysates was assayed before
and every 24 h after inoculation of yeast cells for 5 days. Two mL of
the sample was drawn aseptically with sterile syringe for sugar and
ethanol assay. The ethanol yield was analysed with gas chromatography (Chemito GC 7610) using 10% SE 30 column. The oven
temperature was maintained at 100  C, injection temperature at
130  C with FID detector maintained at 200  C. Nitrogen was used
as carrier gas with a ow rate of 1.20 bar pressure at constant
throughout the analysis.
3. Results and discussion
In this study, two different spent biomasses were used for
ethanol production. One is from industrial spent biomass obtained
after alginate extraction from brown seaweeds. The second is from
RS biomass obtained after extraction of phycobiliproteins. Both the
spent biomass differ in their sugar content because maximum
amount of alginate and other sugars were already extracted from
the BS which was obtained from industry whereas RS was rich in
agar obtained after pigment extraction (agar was not extracted).
Hence, the sugar present in the RS is more than BS.
3.1. Composition of proximate, ultimate and bio-chemicals present
in spent biomass
The proximate analyses of BS and RS biomasses had ash content
of 15.2% and 20.1%, volatile matter of 72.8% and 76.2%, xed carbon
12% and 3.7%, moisture content 8% and 17.2%, respectively, on a dry
weight basis. Reith et al. and Philippsen et al. [49,50] reported the
ash content of farmed brown seaweeds was around 24% DW and
whereas Hong et al. [27], reported 28.6% and 20.6% DW on an
average of ve different brown seaweeds and red seaweed whole
biomass, but in this study the ash content was very less in BS than
RS. However, Francavilla et al. [41], used Gracilaria gracilis for

M.P. Sudhakar et al. / Biomass and Bioenergy 90 (2016) 148e154

from RS. Meinita et al. [30] reported that 10% w/v of substrate
concentration was found suitable for acid hydrolysis at 130  C for
15 min and Cho et al. [24] also reported 13% w/v in 75 mM sulfuric
acid at 121  C for 60 min, but in the present study, only 8% w/v of
substrate was found suitable for acid hydrolysis and yielded a
maximum amount of reducing sugar (13.07 0.008 mg g
1) at
121  C for 20 min (Tables 1 and 2). The 1% H2SO4 treated spent
extract was analysed in FT-Raman along with standard substrates
such as agar, D-galactose and alginic acid. The FT-Raman spectrum
of acid treated hydrolysate of RS biomass showed bands at 748 and
781 cm
1 similar to the spectrum of commercial agar which
showed bands at 740 and 771 cm
1 and standard D-galactose at
764 cm
1 (Fig. S1 in supplementary material) representing skeletal
bending of the galactose ring. The study corroborates with the
ndings of Pereira et al. [51] conrming the presence of galactose
units. The spectral studies of commercial alginic acid showed bands
at (Fig. S2 in supplementary material) 946 cm
1 for O
eH deformation, 1025 cm
1 and 1070 cm
1 for guluronic units, 1105 cm
1
for mannuronic units and 1409 cm
1 for CH2 groups deformation;
Similar bands were observed in 948, 1033, 1092, 1112 and
1397 cm
1 for acid treated hydrolysate of BS (Table 3) conrming
the presence of guluronic and mannuronic which was in agreement
with the study of Pereira et al. [51].

Table 1
Optimization of seaweed spent biomass pretreatment using H2SO4.
Concentration of H2SO4 (v/v) (%)

Total sugar (mg g
1)

Brown seaweed spent
a

Red seaweed spent

65.95 2.92a
96.15 5.49b
129.85 10.23c

62.62 4.36
65.92 8.05a
81.67 3.30b

0.1
0.5
1

Values are expressed as the mean SD (n 3). Values in the same column having
the same letter are not signicant (P < 0.05).

pigment extraction using cascade approach and the left out residue
was used for pyrolysis for energy production where the ash content
was reported to be 20.88% DW. The ash content (15.2% and 20.1%)
obtained in this study was found to be accordance with Francavilla
et al. [41].
The ultimate analyses of BS and RS biomasses resulted in 33.11%
and 32.80% of C, 3.65% and 5% of H, 1.53% and 1.08% of N, 2.44% and
1.82% of S, respectively, on a dry weight basis. Francavilla et al. [41],
reported that Gracilaria gracilis residue (after extraction of phycobiliproteins) contained 31.67% of C, 5.17% of H, 3.98% of N and 1.58%
of S. The mild acid extraction process (hydrolysis of complex sugar)
seems to not affect the algal biomass (BS and RS) composition in
terms of C, H, N, S values and it is in accordance with the composition reported by Francavilla et al. [41]. Total organic carbon found
in BS and RS biomasses were 16.24% and 16.17%, respectively, on a
dry weight basis. The total sugar in BS and RS biomasses was found
to be 136.28 30.09 mg g
1 and 190.71 30.67 mg g
1 (dry
weight), respectively. Total protein content in BS and RS biomasses
was observed to be 1.56 0.06 and 11.076 0.117 mg g
1 (dry
weight), respectively. Total phenol content recorded in spent biomasses of BS and RS was 1.90 0.002 mg g
1 and
1.62 0.004 mg g
1 (dry weight), respectively.

3.3. Fermentation and ethanol yield

Two different seaweeds spent biomass were used for fermentation with baker's yeast to produce ethanol. The reducing sugar
and ethanol yield were calculated every 24 h for a period of 5 days.
The initial reducing sugar concentration of BS (industrial spent
after phycocolloids extraction) and RS (after phycobiliproteins
extraction, no phycocolloid extracted) were observed to be
0.017 0.001% (w/w) and 0.18 0.002% (w/w), respectively. The
gradual decrease of sugar and increase of ethanol was observed
during analysis (Fig. 1). The ethanol yield obtained in BS was
observed to be 0.011% (w/w) and RS was 0.02 0.003% (w/w) on
day 4. The initial reducing sugar of standard substrate such as YPD
and D-galactose was 0.76% and 0.75% (w/w), respectively. The yield
of ethanol with positive control (YPD) was 0.42 0.03% (w/w) and
D-galactose was 0.37 0.04% (w/w) on day 4. The ethanol yield of
RS was observed to be more than BS owing to the extraction of
phycobiliproteins and the rest of the biomass was used for ethanol
production, whereas in the case of BS, phycocolloids were extracted
before fermentation resulting in low yield of ethanol. Standard
substrate showed better production of ethanol by baker's yeast.
This shows that galactose present in the red seaweeds is found be a
good source for bioethanol production. But in case of BS, the industrial spent having less monosugar also can be a potential source
for ethanol production. The sugar which is present in the spent
biomass was responsible for ethanol yield and it depends on the
pretreatment of spent biomass for high sugar yield. Hydrolysis with
commercial enzymes may increase the yield of monosugars from
seaweed spent biomass. In this study, only mild acid pretreatment

3.2. Analysis of seaweed spent hydrolysates

Different concentrations of H2SO4 were used for pre-treatment
of spent biomass. In this study, a concentration of 1% H2SO4 yielded more sugar in BS (81.67 3.30 mg g
1) and RS
(129.85 10.23 mg g
1). Among the different concentrations of
H2SO4 used for substrate optimization in hydrolysis of BS biomass,
the biomass (g) and volume (mL) ratio of 2:50 and 4:50 showed the
maximum amount of reducing sugar yield. The same was obtained

Table 2
Optimization of brown seaweed spent biomass concentration in pretreatment.
Concentration (w/v)

Total sugar (mg g
1)

2:50
4:50
6:50
8:50
10:50

169.39
139.02
108.84
94.49
65.35

Reducing sugar (mg g
1)

0.04
0.04
0.02
0.18
0.33

12.26
13.07
4.74
4.75
4.56

151

0.003
0.008
0.006
0.008
0.005

Table 3
FT-Raman spectra of standards substrates and spent biomass.
Wave number (cm
1)
Band number

10

Agar
D-Galactose
RS
Alginic acid
BS

740.8
703.3
748.5
735.3
699.4

771.9
764.3
781.6
e
787.6

843.7
828.9
827.6
803.3
813.4

891.5
888.21
892.5
877.9
859.1

937.5
956.3
927.7
946.3
948.1

1080.4
1067.5
1073.4
1025.0
1033.0

1252.5
1247.0
1248.0
1070.2
1092.6

1286.6
1307.02
1280.0
1105.5
1112.9

1413.0
1402.9
e
1301.3
1298.7

1472.2
1487.8
e
1409.8
1397.2

RSe Red seaweed spent biomass, BSe Brown seaweed spent biomass.

152

M.P. Sudhakar et al. / Biomass and Bioenergy 90 (2016) 148e154

YPD
1.0

D-Galactose

0.8

Red. sugar
% EtOH

1.0

0.8

Red. sugar
% EtOH

0.9

0.6

0.8

0.8

0.6

0.0
0.2

0.6

0.4

0.5
0.4

0.2

g/g EtOH

0.2

0.4

g/g Red. sugar

0.4

0.6

g/g EtOH

g/g Red. sugar

0.7

0.3
0.2

0.0

0.1

-0.2
0.0

0.0

-1

-0.2
-1

Days

Days

Red seaweed spent

0.20

Brown seaweed spent

0.03

0.03

0.03

Red. sugar
% EtOH

0.01

0.10

0.00
-1

0.02

0.02

0.01

0.01

g/g EtOH

0.15

g/g Red. Sugar

0.02

g/g EtOH

g/g Red. Sugar

Red. sugar
% EtOH

0.00

0.00
-1

Days

Days

Fig. 1. Reducing sugar depletion and ethanol yield from standard substrates (YPD and D-galactose) and seaweed spent biomass (red and brown).

showed better results with industrial spent BS. Since RS contain

polysaccharide such as agar, they need proper hydrolysis such as
enzymatic, bacterial and/or high acid concentration ratio to convert

complex sugars to simple monosugars for high ethanol yield. Increase in concentration of acid may lead to produce more HMF
(Hydroxymethyl Furfural) than simple sugars such as galactose or

Table 4
Comparison of ethanol yield from various seaweed feedstocks.
Seaweeds
Red seaweeds
Gracilaria verrucosa
Kappaphycus alvarezii (cottonii)
Gelidium amansii
Gracilaria chorda
Gracilaria tenuistipitata
Kappaphycus alvarezii
E. cottonii
Gelidium elegans
Gracilaria salicornia
Kappaphycus alvarezii
Brown seaweeds
Laminaria hyperborea
Undaria pinnatida
Alaria Crassifolia
Saccharina latissima
Saccharina japonica
Sargassum spp.
Green seaweeds
Floating residue (FR)
Ulva pertusa
Ulva fasciata
Ulva lactuca
Chaetomorpha linum
Gracilaria corticata spent (RS)
Industrial spent biomass (BS)

Biomass type

Microorganism

Country

Ethanol yield

Reference

Residual agar pulp

Whole biomass
Whole biomass
Whole biomass
Whole biomass
Spent biomass
Seaweed solid wastes
Whole biomass
Whole biomass
Whole biomass

Saccharomyces cerevisiae HAU strain

Commercial brewer's yeast
Commercial Saccharomyces cerevisiae
Commercial Saccharomyces cerevisiae
Commercial Saccharomyces cerevisiae
Saccharomyces cerevisiae (NCIM 3523)
S. cerevisiae (YSC2, type II)
Saccharomyces cerevisiae IAM 4178
E. coli KO11
Saccharomyces cerevisiae CBS1782

India
Indonesian
Vietnam
Vietnam
Vietnam
Mandapam, India
Malaysia
Japan
China
Brazil

14.89 g L
1
1.7 g L
1
0.83 g L
1
0.5 g L
1
0.6 g L
1
2.06%
0.27 g/g
55.0 g L
1
7.9%
38e64.3 g L
1

[21]
[29]
[30]
[30]
[30]
[52]
[53]
[54]
[55]
[56]

Whole biomass
Discarded as waste
Whole biomass
Whole biomass
Whole biomass
Whole biomass

Pichia angophorae CBS 5830

Pichia angophorae KCTC 17574
Saccharomyces cerevisiae IAM 4178
Saccharomyces cerevisiae
Pichia angophorae KCTC 17574
Saccharomyces cerevisiae

Norway
Korea
Japan
UK
Korea
Philippines

0.43 g g
1
9.42 g L
1
34.4 g L
1
0.45%
7.7 g L
1
2.79 g L
1

[23]
[24]
[54]
[57]
[58]
[59]

Industrial spent
Whole biomass
Whole biomass
Whole biomass
Whole biomass
Pigment extracted spent
Alginate extracted spent

Saccharomyces cerevisiae
Saccharomyces cerevisiae IAM 4178
Saccharomyces cerevisiae (MTCC 180)
Clostridium beijerinckii
S. cerevisiae ATCC 96581
Baker's yeast
Baker's yeast

China
Japan
India
Netherland
Denmark
India
India

0.143 L kg
1 FR
30.0 g L
1
0.45 g/g
0.3 g L
1
15% w/w
3.02 g L
1
1.61 g L
1

[5]
[54]
[60]
[61]
[62]
Present study
Present study

M.P. Sudhakar et al. / Biomass and Bioenergy 90 (2016) 148e154

cellulose. These HMFs are inhibitory to microorganisms [28].

Comparison of ethanol yield from various seaweed substrates was
represented in Table 4.
4. Conclusion
For the past few years, vast research was conducted on ethanol
production from seaweeds using various yeasts and bacteria for
hydrolysis, and using enzymes such as cellulase etc. for direct
conversion of seaweeds to fermentable substrate. The present
study revealed the easy way of conversion of seaweed spent to
fermentable sugars for ethanol production and effective utilization
of seaweed spent biomass from seaweed industry. The RS was
inferred to be a better producer of ethanol than the BS. Cultivation
of these seaweeds in India and direct utilization of seaweed as such
will help resolve the energy demand in the future. Moreover,
identication of potential bacterial strain for hydrolysing seaweed
polysaccharide and co-cultivation of yeast would enhance the
production of ethanol which will also be a challenging task. Further
study will help to scale-up the upstream and downstream process
of ethanol for industrial spent.
Acknowledgements
The authors thank Department of Science and Technology, New
Delhi for nancial support (Ref. No. DST/TSG/AF/2010/12) and Shri
AMM Murugappa Chettiar Research Centre for providing the laboratory facilities. We also thank SAIF-STIC, Cochin University of
Science and Technology, Kerala and SAIF-IIT Madras for sample
analysis. Thanks to Dr. R. Jayakumar for his help in literature
collection. Special thanks to Dr. Valsaraj and Dr. B. Annapurna for
their help in language editing of the manuscript.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biombioe.2016.03.031.
References
[1] L. Brennan, P. Owende, Biofuels from micro-algaee a review of technologies
for production, processing, and extractions of biofuels and co-products,
Renew. Sustain. Energy Rev. 14 (2010) 557e577.
[2] P.S. Nigam, A. Singh, Production of liquid biofuels from renewable resources,
Prog. Energy Combust. Sci. 37 (2011) 52e68.
[3] R.P. John, G.S. Anisha, K.M. Nampoothiri, A. Pandey, Micro and macroalgal
biomass: a renewable source for bioethanol, Bioresour. Technol. 102 (2011)
186e193.
[4] Y.S. Hong, H.H. Yoon, Ethanol production from food residues, Biomass Bioenergy 35 (2011) 3271e3275.
[5] L. Ge, P. Wang, H. Mou, Study on saccharication techniques of seaweed
wastes for the transformation of ethanol, Renew. Energy 36 (2011) 84e89.
[6] S. Prasad, A. Singh, H.C. Joshi, Ethanol as an alternative fuel from agricultural,
industrial and urban residues, Resour. Conserv. Recycl. 50 (2007) 1e39.
[7] A. Manilal, S. Sujith, G.S. Kiran, J. Selvin, C. Shakir, R. Gandhimathi,
M.V.N. Panikkar, Biopotentials of seaweeds collected from southwest coast of
India, J. Mar. Sci. Tech. 17 (2009) 67e73.
[8] Y. Li, S. Kim, Utilization of seaweed derived ingredients as potential antioxidants and functional ingredients in the food industry: an overview, Food Sci.
Biotechnol. 20 (2011) 1461e1466.
[9] K. Chojnacka, A. Saeid, Z. Witkowska, L. Tuhy, Biologically active compounds
in seaweed extracts - the prospects for the application, Open Conf. Proc. J. 3
(Suppl. 1-M4) (2012) 20e28.
[10] P.V. Subba Rao, V.A. Mantri, Indian seaweed resources and sustainable utilization: scenario at the dawn of a new century, Curr. Sci. 91 (2006) 164e174.
[11] FAO, Fishery Statistical Collections Global Aquaculture Production, 2010.
http://www.fao.org/shery/statistics/global-aquaculture-production/en
(accessed 24.09.10).
[12] B.H. Buck, C.M. Buchholz, The offshore-ring: a new system design for the open
ocean aquaculture of macroalgae, J. Appl. Phycol. 16 (2004) 355e368.
[13] S. Kraan, M.D. Guiry, The seaweed resources of Ireland, in: A. Critchley,
M. Ohno, D. Largo (Eds.), Seaweed Resources of the World, a CD-Rom Project,

153

ETI, Amsterdam, 2006.

[14] R. Kaas, The seaweed resources of France, in: A.T. Critchly, M. Ohno, D.B. Largo
(Eds.), The Seaweed Resources of the World. Interactive CD ROM, ETI, The
Netherlands, 2006.
[15] M. Ohno, D.B. Largo, The seaweed resources of Japan, in: A.T. Critchly,
M. Ohno, D.B. Largo (Eds.), The Seaweed Resources of the World. Interactive
CD ROM, ETI, The Netherlands, 2006.
[16] C.Y. Wu, S.J. Pang, The seaweed resources of China, in: A.T. Critchly, M. Ohno,
D.B. Largo (Eds.), The Seaweed Resources of the World. Interactive CD ROM,
ETI, The Netherlands, 2006.
[17] K.A. Jung, S. Lim, Y. Kim, J.M. Park, Potentials of macroalgae as feedstocks for
biorenery, Bioresour. Technol. 135 (2013) 182e190.
[18] R.M. Oza, V. Krishnamurthy, Studies on carposporic rhythm of Gracilaria
verrucosa (Huds) Papenf, Bot 11 (Mar 1968) 118e121.
[19] R.M. Oza, Studies on Indian Gracilaria V. Seasonal variation in agar and gel
strength of Gracilaria corticata, J. Ag. Occur. Coast Veraval. Bot. 21 (Mar 1978)
165e167.
[20] C.S. Goh, K.T. Lee, A visionary and conceptual macroalgae-based third generation bioethanol (TGB) biorenery in Sabah, Malaysia as an underlay for
renewable and sustainable development, Renew. Sustain. Energy Rev. 14
(2010) 842e848.
[21] J.M. Adams, T.A. Toop, J.M. Gallagher, I.S. Donnison, Seasonal variation in
Laminaria digitata and its impact on biochemical conversion routes to biofuels,
Bioresour. Technol. 102 (2011) 9976e9984.
[22] S. Kumar, R. Gupta, G. Kumar, D. Sahoo, R.C. Kuhad, Bioethanol production
from Gracilaria verrucosa, a red alga, in a biorenery approach, Bioresour.
Technol. 135 (2013) 150e156.
[23] S.J. Horn, I.M. Aasen, K. stgaard, Ethanol production from seaweed extract,
J. Ind. Microbiol. Biot. 25 (2000) 249e254.
[24] Y. Cho, H. Kim, S. Kim, Bioethanol production from brown seaweed, Undaria
pinnatida, using NaCl acclimated yeast, Bioprocess Biosyst. Eng. 36 (2013)
713e719.
[25] P. Lenihan, A. Orozco, E. O'Neill, M.N.M. Ahmad, D.W. Rooney, G.M. Walker,
Dilute acid hydrolysis of lignocellulosic biomass, Chem. Eng. J. 156 (2010)
395e403.
[26] G.T. Jeong, S.K. Kim, D.H. Park, Application of solid-acid catalyst and marine
macro-algae Gracilaria verrucosa to production of fermentable sugars, Bioresour. Technol. 181 (2015) 1e6.
[27] I.K. Hong, H. Jeon, S.B. Lee, Comparison of red, brown and green seaweeds on
enzymatic saccharication process, J. Ind. Eng. Chem. 20 (2014) 2687e2691.
Progress in bioethanol processing, Prog. Energy
[28] M. Balat, H. Balat, C. Oz,
Combust. Sci. 34 (2008) 551e573.
[29] M.D.N. Meinita, J. Kang, G. Jeong, H.M. Koo, S.M. Park, Y. Hong, Bioethanol
production from the acid hydrolysate of the carrageenophyte Kappaphycus
alvarezii (cottonii), J. Appl. Phycol. 24 (2012) 857e862.
[30] M.D.N. Meinita, B. Marhaeni, T. Winanto, G. Jeong, M.N.A. Khan, Y. Hong,
Comparison of agarophytes (Gelidium, Gracilaria, and Gracilariopsis) as potential resources for bioethanol production, J. Appl. Phycol. 25 (2013)
1957e1961.
[31] S.W. Kim, C.H. Hong, S.W. Jeon, H.J. Shin, High-yield production of biosugars
from Gracilaria verrucosa by acid and enzymatic hydrolysis processes, Bioresour. Technol. 196 (2015) 634e641.
[32] J.L. Fortman, S. Chhabra, A. Mukhopadhyay, H. Chou, T.S. Lee, E. Steen,
J.D. Keasling, Biofuels alternatives to ethanol: pumping the microbial well,
Trends Biotechnol. 26 (2008) 375e381.
[33] S.K. Lee, H. Chou, T.S. Ham, T.S. Lee, J.D. Keasling, Metabolic engineering of
microorganisms for biofuels production: from bugs to synthetic biology to
fuels, Curr. Opin. Biotechnol. 19 (2008) 556e563.
[34] S. Kraan, Mass-cultivation of carbohydrate rich macroalgae, a possible solution for sustainable biofuel production, Mitig. Adapt Strateg. Glob. Change
(2010), http://dx.doi.org/10.1007/s11027-010-9275-5.
[35] M. Daroch, S. Geng, G. Wang, Recent advances in liquid biofuel production
from algal feedstocks, Appl. Energy 102 (2013) 1371e1381.
[36] RFA (Renewable Fuels Association), The Gallagher Review of the Indirect Effects of Biofuels Production, 2008. Available from: http://www.
renewablefuelsagency.gov.uk/_db/_documents/Report_of_the_Gallagher_
review.pdf.
[37] F.O. Licht. World Ethanol and Biofuels Report, 2009; 7(18): 365.
[38] GRFA, Global Renewable Fuels Alliance, 2010. http://www.globalrfa.org
(accessed 08.05.10).
[39] M. Kumar, V. Gupta, N. Trivedi, P. Kumari, A.J. Bijo, C.R.K. Reddy, B. Jha,
Desiccation induced oxidative stress and its biochemical responses in intertidal red alga Gracilaria corticata (Gracilariales, Rhodophyta), Environ. Exp.
Bot. 72 (2011) 194e201.
[40] M.P. Sudhakar, A. Jagatheesan, K. Perumal, K. Arunkumar, Methods of phycobiliprotein extraction from Gracilaria crassa and its applications in food
colourants, Algal Res. 8 (2015) 115e120.
[41] M. Francavilla, P. Manara, P. Kamaterou, M. Monteleone, A. Zabaniotou,
Cascade approach of red macroalgae Gracilaria gracilis sustainable valorization
by extraction of phycobiliproteins and pyrolysis of residue, Bioresour. Technol. 184 (2015) 305e313.
[42] ASTM Standard E1705, Standard Technology Relating to Biotechnology, ASTM
International, 1995.
[43] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric
method for determination of sugars and related substances, Anal. Chem. 28

154

M.P. Sudhakar et al. / Biomass and Bioenergy 90 (2016) 148e154

(1956) 350e356.
[44] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing
sugars, Anal. Chem. 31 (1972) 426e428.
[45] M.S. Taga, E.E. Miller, D.E. Pratt, Chia seeds as a source of natural lipid antioxidants, J. Am. Oil Chem. Soc. 61 (1984) 928e931.
[46] E. Barbarino, S.O. Lourenco, An evaluation of methods for extraction and
quantication of protein from marine macro and micro algae, J. Appl. Phycol.
17 (2005) 447e460.
[47] O.H. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement
with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265e275.
[48] M.D.N. Meinita, Y. Hong, G. Jeong, Comparison of sulfuric and hydrochloric
acids as catalysts in hydrolysis of Kappaphycus alvarezii (cottonii), Bioproc.
Biosyst. Eng. 35 (2012) 123e128.
[49] J.H. Reith, E.P. Deurwaarder, K. Hemmes, A. Curvers, P. Kamermans,
W. Brandenburg, et al., Bio-offshore:grootschalige teelt van zeewieren in
combinative metoffshore Windparken in de Noordzee, Energy Research
Centre of the Netherlands, Dutch, 2005.
[50] A. Philippsen, P. Wild, A. Rowe, Energy input, carbon intensity and cost for
ethanol produced from farmed seaweed, Renew. Sustain. Energy Rev. 38
(2014) 609e623.
[51] L. Pereira, A. Sousa, H. Coelho, A.M. Amado, P.J.A. Ribeiro-Claro, Use of FTIR,
FT-Raman and 13C-NMR spectroscopy for identication of some seaweed
phycocolloids, Biomol. Eng. 20 (2003) 223e228.
[52] Y. Khambhaty, K. Mody, M.R. Gandhi, S. Thampy, P. Maiti, H. Brahmbhatt,
K. Eswaran, P.K. Ghosh, Kappaphycus alvarezii as a source of bioethanol, Bioresour. Technol. 103 (2012) 180e185.
[53] I.S. Tan, K.T. Lee, Enzymatic hydrolysis and fermentation of seaweed solid
wastes for bioethanol production: an optimization study, Energy (2014) 1e10.

[54] M. Yanagisawa, K. Nakamura, O. Ariga, K. Nakasaki, Production of high concentrations of bioethanol from seaweeds that contain easily hydrolyzable
polysaccharides, Process Biochem. 46 (2011) 2111e2116.
[55] X. Wang, X. Liu, G. Wang, Two-stage hydrolysis of invasive algal feedstock for
ethanol fermentation, J. Integr. Plant Biol. 53 (2011) 246e252.
[56] P.I. Hargreaves, C.A. Barcelos, A.C. Augusto da Costa, N. Pereira Jr., Production
of ethanol 3G from Kappaphycus alvarezii: evaluation of different process
strategies, Bioresour. Technol. 134 (2013) 257e263.
[57] J.M. Adams, J.A. Gallagher, I.S. Donnison, Fermentation study on Saccharina
latissima for bioethanol production considering variable pre-treatments,
J. Appl. Phycol. 21 (2009) 569e574.
[58] J. Jang, Y. Cho, G. Jeong, S. Kim, Optimization of saccharication and ethanol
production by simultaneous saccharication and fermentation (SSF) from
seaweed, Saccharina japonica, Bioprocess Biosyst. Eng. 35 (2012) 11e18.
[59] M.G. Borines, R.L. de Leon, J.L. Cuello, Bioethanol production from the macroalgae Sargassum spp, Bioresour. Technol. 138 (2013) 22e29.
[60] N. Trivedi, C.R.K. Reddy, R. Radulovich, B. Jha, Solid state fermentation (SSF)derived cellulase for saccharication of the green seaweed Ulva for bioethanol
production, Algal Res. 9 (2015) 48e54.
[61] H. Wal, B.L.H.M. Sperber, B. Houweling-Tan, R.R.C. Bakker, W. Brandenburg,
pez-Contreras, Production of acetone, butanol, and ethanol from
A.M. Lo
biomass of the green seaweed Ulva lactuca, Bioresour. Technol. 128 (2013)
431e437.
[62] N. Schultz-Jensen, A. Thygesen, F. Leipold, S.T. Thomsen, C. Roslander,
H. Lilholt, A.B. Bjerre, Pretreatment of the macroalgae Chaetomorpha linum
for the production of bioethanol e comparison of ve pretreatment technologies, Bioresour. Technol. 140 (2013) 36e42.