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Bioresource Technology 150 (2013) 106112

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Enzymatic hydrolysis and production of bioethanol from common

macrophytic green alga Ulva fasciata Delile
Nitin Trivedi a,b, Vishal Gupta a, C.R.K. Reddy a,b,, Bhavanath Jha a
a
b

Discipline of Marine Biotechnology and Ecology, CSIR Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India
Academy of Scientic and Innovative Research (AcSIR-CSMCRI), Bhavnagar 364002, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Green seaweed Ulva fasciata was used

as a feedstock for bioethanol

production.
 Enzyme reusability conrmed for two
successive saccharication cycles.
 Ethanol yield obtained was 0.45 g/g
reducing sugar accounting 88.2%
efciency.
 Dispenses the addition of yeast
extract and peptone in the
fermentation medium.

a r t i c l e

i n f o

Article history:
Received 17 July 2013
Received in revised form 20 September
2013
Accepted 25 September 2013
Available online 2 October 2013
Keywords:
Bioethanol
Cellulose
Fermentation
Macroalgae

a b s t r a c t
The green seaweed Ulva which proliferates fast and occurs abundantly worldwide was used as a feedstock for production of ethanol following enzymatic hydrolysis. Among the different cellulases investigated for efcient saccharication, cellulase 22119 showed the highest conversion efciency of
biomass into reducing sugars than Viscozyme L, Cellulase 22086 and 22128. Pre-heat treatment of biomass in aqueous medium at 120 C for 1 h followed by incubation in 2% (v/v) enzyme for 36 h at 45 C
gave a maximum yield of sugar 206.82 14.96 mg/g. The fermentation of hydrolysate gave ethanol yield
of 0.45 g/g reducing sugar accounting for 88.2% conversion efciency. These values are substantially
higher than those of reported so far for both agarophytes and carrageenophytes. It was also conrmed
that enzyme can be used twice without compromising on the saccharication efciency. The ndings
of this study reveal that Ulva can be a potential feedstock for bioethanol production.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
The growing concern on depletion of fossil fuels and their environmental effects of burning, particularly greenhouse gas emissions, have led to search for viable renewable fuel alternatives
(Hahn-Hgerdal et al., 2006). Plant biomass is the only renewable
source capable of producing alternative transport liquid fuels.
Corresponding author at: Discipline of Marine Biotechnology and Ecology, CSIRCentral Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India.
Tel.: +91 278 256 5801/256 3805x6140; fax: + 91 278 256 6970/256 7562.
E-mail address: crk@csmcri.org (C.R.K. Reddy).
0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.09.103

The most widely used transport biofuel around the world is bioethanol which showed a signicant increase in production from less
than a billion liters in 1975 to more than 86 billion liters in 2010
and is expected to reach 100 billion liters by 2015 (Licht, 2006).
Bioethanol is largely derived from starch/cellulose based biomass
i.e. plants, crops and agricultural-wastes. Currently, Brazil, U.S.
and Canada are the leaders in the production of bioethanol from
two major sources namely corn and sugarcane (Chiaramonti,
2007). The high cost involved in production of ethanol from
sugarcane and the food vs fuel debate over corn ethanol led to
search for new feedstock. Subsequently, agriculture waste rich in
ligno-cellulose has been considered as potential alternative

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N. Trivedi et al. / Bioresource Technology 150 (2013) 106112

feedstock. Fast-growing short-rotation forest trees such as Populus

and Eucalyptus having large amounts of cellulose-rich biomass
emerged as promising source for bioenergy and biopolymer production. However, lignocellulosic materials are hard to depolymerise because of the presence of complex lignin and hemicelluloses
over cellulose. Different chemical pre-treatment methods are employed to increase cellulose accessibility (Girio et al., 2010). Therefore production of fermentable sugars from lignocellulosic biomass
tends to be complex, capital intensive and also has associated
inherent environmental concerns.
Recently, marine macroalgal species have gained considerable
global attention as source of third generation biofuels (Horn
et al., 2000; Goh and Lee, 2010; Wang et al., 2011; Khambhaty
et al., 2012; Meinita et al., 2012). The major advantages offered
by seaweeds over terrestrial biomass are (1) higher biomass production rate per unit area, (2) do not compete with agricultural
plants for land, (3) require no agricultural input such as fertilizer,
pesticides and water, and (4) easier depolymerisation as it does
not contain lignin in their cell wall (Jones and Mayeld, 2012).
So far, bioethanol production from seaweeds has been mainly
conned to a few phycocolloid yielding species belonging to the
genera of Kappaphycus, Gelidium, Gracilaria, Sargassum and
Laminaria. This strategy would not only affect the existing multibillion hydrocolloid industry but also may lead to another new debate
Hydrocolloid vs Fuel. Alternatively, Kumar et al. (2013) demonstrated production of bioethanol from cellulosic residue following
the extraction of hydrocolloid from seaweed biomass. Nevertheless, the lower cellulose content of residue may prevent it from
being a viable feedstock option considering the growing demand
for bioethanol. Therefore selection of seaweed species with higher
cellulose content together with higher growth rate is of paramount
importance for sustainable bioethanol production.
It in this context, a green seaweed Ulva fasciata Delile was selected as a potential feedstock for bioethanol production following
enzymatic hydrolysis. This species besides having higher polysaccharide content grows luxuriantly and has worldwide distribution
regardless of geographical barriers. This species is also regarded as
an opportunistic species with potentials to form blooms (green
tide) when there is a sudden outburst of nutrients in the aquatic
streams.
2. Methods
2.1. Collection of algal sample
The green seaweed U. fasciata Delile (Chlorophyceae) was
collected from Veraval (N 20 54.870 , E 70 20.830 ) coast of Gujarat,
India. The Seaweed samples were washed thoroughly with tap
water to remove salts, epiphytes and debris and dried to a constant
weight at temperature of 50 C. After drying, the seaweed samples
were powdered using grinder for chemical composition analysis.
2.2. Chemical composition of U. fasciata
Selection of biomass is crucial in terms of techno-economic aspects. Therefore collected algal sample was rstly characterized for
its growth rate and proximate chemical composition. For growth
rate determination, fresh algal samples were rst acclimatized to
laboratory condition by culturing in enriched seawater medium
(Provasoli, 1968) at temperature of 25 1 C. The growth rate of
the alga was thereafter determined as daily growth rate or DGR
(%). For DGR (%) calculation, algal thallus weighing 100 mg was
cultured in 500 mL round at bottom aerated asks containing
400 mL sterile seawater supplemented with PES medium for
15 days at temperature 25 1 C under white cool uorescent light

107

of irradiance 50 lmol photons m2 s1 under day night photoperiod of light:dark 12:12 h. During this culture duration medium
was changed every third day. The DGR (%) was calculated by estimating the weight of each thalli at every third day following the
equation DGR % W t =W 0 1=t  1  100 where Wt is the weight
after time t and W0 is the initial weight at time t = 0.
The chemical composition analysis of U. fasciata includes the
determination of protein, carbohydrate, lipid and cellulose content
following the method of Lowry et al. (1951), Dubois et al. (1956),
Bligh and Dyer (1959) and Mihranyan et al. (2004), respectively.
The major structural groups of extracted cellulose were characterized using Fourier transform infrared (FT-IR) spectroscopy. The
FT-IR spectrum of the cellulose was obtained on Perkin-Elmer
spectrum GX FT-IR system (Perkin-Elmer, USA) and was recorded
in the absorption mode in the range 4000400 cm1. Total elemental (CHNS) content was determined using CHN Elemental Analyzer
(Perkin-Elmer Model 2400, USA), calibrated with acetanilide as a
reference standard. Moisture content of alga was also determined
using analytical methods established by the National Renewable
Energy Laboratory (NREL) (Sluiter et al., 2008). Ash content was
determined by weighing samples before and after heating in a furnace at temperature of 550 C for 12 h.
2.3. Optimization of enzymatic hydrolysis of algal biomass
The optimization of enzymatic hydrolysis of biomass was carried out for enzyme dosage, incubation period and liquid: biomass
ratio. Commercial cellulase enzyme (Viscozyme L) procured from
Novozyme, Denmark was employed for biomass hydrolysis. Dried
algal powder (1 g) was hydrolyzed with different concentration
of cellulase from 1%, 2% and 5% v/v in a x volume (20 mL) of sodium acetate buffer (pH 4.8) and incubated for different time interval from 6 to 42 h at 45 C on an orbital shaker with a speed of
150 rpm. Samples were taken out periodically after an interval of
6 h each and centrifuged. The reducing sugar was measured spectrophotometrically using 3,5-dinitrosalisylic acid (DNS) method
(Miller, 1959). After optimization of the enzyme dosage and incubation period, biomass to liquid volume (sodium acetate buffer, pH
4.8) ratio was optimized. For this, dried algal powder (5 g) was
hydrolyzed with optimum enzyme dosage of 2% (v/v) in different
volumes of sodium acetate buffer (pH 4.8) ranging from 25 to
100 mL with increments of 25 mL units. After hydrolysis at
optimized conditions, reducing sugars was measured using DNS
method.
After optimization of hydrolysis conditions, effect of different
pretreatments such as dilute acid (H2SO4), liquid ammonia and
heat treatment to biomass with and without aqueous buffer were
investigated on reducing sugars yield. The acid pretreatment
include treatment of algal biomass with different concentrations
of H2SO4 i.e. 0.1, 0.5 and 1% (v/v) for 1 h at 100 C. The liquid
ammonia pretreatment was performed at different concentrations
of 1, 2 and 4% (v/v) for different time periods of 15, 30 and 60 min.
The heat treatment was given to algal biomass with and without
aqueous buffer in autoclave at a temperature of 120 C and pressure 15 psi for 30, 60 and 90 min. The acid and liquid ammonia
pretreatments were followed by rinsing of biomass with distilled
water till neutrality. After each pretreatment, biomass was hydrolysed enzymatically under the conditions optimized earlier.
After optimization of pretreatment conditions, the hydrolyzing
efciencies of different commercial cellulases (Novozyme, Denmark) such as Cellulase 22128, Cellulase 22119, and Cellulase
22086 were also investigated. Saccharication efciencies of these
enzymes were determined under optimized conditions for different incubation periods ranging from 12 to 48 h with increments
of 12 h. Among the different enzyme tested, the enzyme shown
higher saccharication efciency was selected for further studies.

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N. Trivedi et al. / Bioresource Technology 150 (2013) 106112

This study further investigated the reusability of the enzyme

showing optimum results in terms of reducing sugar yields on
hydrolysis. For this, 10 g of dry algal biomass was employed for
pretreatment followed by enzymatic hydrolysis under optimized
conditions. The hydrolyzed solution was centrifuged at
12,000 rpm for 20 min and supernatant was collected. Some reduction in liquid volume was recorded therefore the addition of second batch of algal biomass was accordingly adjusted in order to
bring the optimum biomass:liquid volume ratio. Same process
was repeated for third successive saccharication. After every successive hydrolysis the sugar content was determined by DNS
method.
Further to conrm that the sugar yields obtained were from cellulose fraction of algal biomass and not from other algal part, enzymatic hydrolysis of cellulose extracted from the same alga
(U. fasciata) was carried out under optimized conditions in same
manner as did for algal biomass. The quantication of the reducing
sugars after hydrolysis was compared with the yields obtained
after hydrolysis of whole algal biomass.
2.4. Fermentation of the algal hydrolysate
Fermentation of the algal hydrolysate was carried out using
yeast strain Saccharomyces cerevisiae (MTCC No. 180) procured
from Microbial Type Culture Collection and Gene Bank, Institute
of Microbial Technology, Chandigarh, India. S. cerevisiae was maintained as pure culture on YEPD medium (Yeast extract: 0.3%, Peptone: 1%, Dextrose, 2% w/v, pH 6.8). The inoculum was prepared by
inoculating a loop full of yeast cells in sterile YEPD medium followed by incubation at temperature 28 1 C for 24 h on an orbital
shaker at a speed of 120 rpm. The fermentation efciency of the
yeast was determined by the estimation of ethanol yields after
every 12 h of incubation. For ethanol yield determination, 5 mL
of fermentation broth was withdrawn and centrifuged at
10,000 rpm at 4 C. The supernatant obtained was analyzed using
a GCMS (Shimadzu QP 2010) coupled with Shimadzu make (QP
2010) head space (AOC-5000) analyzer (Khambhaty et al., 2012).
The retention time and mass fragmentation were the criterion selected for the conrmation of ethanol production in fermentation
broth. The HPLC grade ethanol was used as a reference standard
for retention time and mass fragmentation determination. This
data was used as a control.
Algal hydrolysate obtained after enzymatic hydrolysis of 15 g
biomass was enriched with peptone (5 g/L) and yeast extract
(3 g/L). The fresh yeast culture (109 CFU/mL) was then inoculated
to fermentation broth. Fermentation was carried out at a temperature of 28 2 C on an orbital shaker with shaking speed of
120 rpm for an incubation period ranging from 12 to 48 h with
increments of 12 h. Samples were withdrawn at regular interval
of 12 h and analyzed for ethanol yield and residual reducing sugars
by GCMS and DNS method, respectively. An additional fermentation experiment was carried out wherein yeast strain was inoculated directly into seaweed hydrolysate obtained from 15 g
biomass without any addition of fermentation medium ingredients
i.e. yeast extract and peptone.
This study also included with the scaling up of the process of
enzymatic hydrolysis of algal biomass and fermentation in order
to realize the ethanol yields. For this, different weights of seaweed
biomass 15, 40 and 100 g dry weight were hydrolyzed and fermented under optimized conditions.

(for sample size of two treatments), ANOVA (for >2 treatments),

and Post-Hoc Tukeys HSD test for comparison of signicant means
using OriginPro 8.5.
3. Results and discussion
3.1. Growth rate of U. fasciata
The plant species having higher growth rate and higher polysaccharide content have been a preferred source of feedstock for sustainable bioethanol production at industrial scale. The growth rate
of U. fasciata investigated in the laboratory conditions at different
temperature regimes from 15 to 30 C showed a linear increase
in DGR (%) from 19.15 0.91 to 24.25 1.62 up to 25 C and thereafter declined sharply to 8.38 3.3 at 30 C (Fig. 1). The red algal
species belonging to the genus Gracilaria and Kappaphycus from
which bioethanol has been reported in the past were reported to
have DGR (%) values in the range of 38% (Mantri et al., 2009; Padhi
et al., 2011) which is remarkably lower than that of U. fasciata
investigated in the present study. Bruhn et al. (2011) while investigating the bioenergy potential of U. lactuca, reported an estimated production of 45 t DW ha1 y1 based on data generated
from land-based experimental cultivation experiments and stated
that this value is 220 times the production potential of conventional terrestrial energy crops such as wheat straw, willow,
miscanthus, or maize and 3 times the production of brown algae
in temperate waters. Fast-growing, short-rotation forest trees, such
as Populus and Eucalyptus have also been considered as potential
feedstock for bio-energy and biopolymer production. But the complexity of ligno-cellulose in such terrestrial energy crops invariably
requires harsh chemical treatments making the process energy
intensive and environmentally hazardous. The higher growth rate
of this alga provided a circumstantial evidence for its utility as potential source of feedstock.
3.2. Chemical composition of U. fasciata
The chemical composition of U. fasciata is summarized in
Table 1. The carbohydrate, protein and lipid content were found
to be 43 4.5%, 14.4 2.2% and 1.83 0.3%, respectively on dry
weight basis. Wal et al. (2012) also reported similar chemical composition in another closely related taxa U. lactuca. Kumar et al.
(2011) while studying the nutritional properties of different tropical seaweeds reported slightly higher yields of carbohydrates ranging from 46% to 57% on dry weight basis in different taxa of Ulva.
Although, the carbohydrate content in red and brown algae has
also been reported to be in the similar range to that of green algal
taxa U. fasciata, they are utilized for gelling applications in diverse
products and forms a basis for multibillion industry worldwide.
Therefore, diversication of these valuable biopolymers for bioethanol production may seriously affect the existing industry and may

2.5. Statistical analysis

All the experiments were conducted in replicates of three and
the results are presented as mean S.D. Further the signicant differences among the mean values were evaluated by students t-test

Fig. 1. Daily growth rate (%) of Ulva fasciata at different temperatures.

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N. Trivedi et al. / Bioresource Technology 150 (2013) 106112

Table 1
Biochemical composition of U. fasciata.
Proximate composition

Relative % on dry weight basis

Carbohydrate
Protein
Lipid
Moisture
Ash
Carbon
Hydrogen
Nitrogen
Sulfer
Cellulose

43 4.5
14.4 2.2
1.83 0
19.86 1.3
16 2.7
25.64 1.6
5.75 2.4
3.13 0.88
5.52 0.45
15 2.3

lead to another debate Fuel vs Hydrocolloids. Recently, Kumar

et al. (2013) demonstrated an integrated approach for recovery
of both hydrocolloid (agar) and cellulose from red algal taxa Gracilaria verrucosa. Incidentally, the cellulosic content of both red and
brown algal taxa has been reported to be low and in general found
to be in the range of 210% on dry weight basis (Siddhanta et al.,
2009). In contrast, the green algal taxa particularly U. fasciata had
cellulosic content as high as 15 2.3% on dry weight basis.
The FT-IR spectrum of cellulose, extracted from U. fasciata, reveals characteristic functional groups (Supplementary Fig. 1). The
FT-IR spectra exhibited specic absorbance of OH stretching at
34313435 cm1, weak CH stretching at 2929 and 2934 cm1,
bound H2O stretching at 16301640, CH bending at 14201422,
COC bending at 10201022 cm1 (Siddhanta et al., 2011). A
stretching of COC, CO at 10001200 cm1 corresponds to presence of carbohydrates. The green seaweed species U. fasciata investigated in this study possess both high cellulose and growth
characteristics thereby this biomass can be a preferred feedstock
for viable bioethanol industry. Additionally this alga has copious
amount of protein (14.4 2.2%) which represent yet another
important utility of this feedstock as a potential source of proteinaceous diet (Wassef et al., 2013).

3.3. Optimization of enzymatic hydrolysis

The saccharication of the alga U. fasciata with the enzyme
Viscozyme L was optimized with respect to enzyme dosage, incubation period and biomass to liquid ratio. Different enzyme dosage
of 1%, 2% and 5% (w/v) resulted into a reducing sugar yield of
48.65 4.3, 159.22 11.9 and 132.69 8.2 mg/g biomass, respectively. The enzyme dosage of 2% (w/v) was found optimum and
was employed for hydrolysis of biomass in subsequent optimization studies.
The optimization of enzymatic hydrolysis period is described in
Fig. 2. The reducing sugar yields were estimated to increase linearly with increase in incubation period from 6 to 36 h and ranged

Fig. 2. Optimization of enzymatic hydrolysis of Ulva fasciata with respect to

different incubation periods.

109

between 72.73 3.2 mg/g and 168.15 6.3 mg/g. Further

increase in incubation period to 42 h showed a decline in reducing
sugar yield to 151.21 2.2 mg/g (Fig. 2). The optimization of
biomass to liquid ratio resulted in an optimum reducing sugar
yield of 184.4 2.25 mg/g when biomass:liquid ratio was adjusted
to 1:20 and the same declined to 164 2.1 mg/g and
136 1.85 mg/g at the biomass:liquid ratio of 1:15 and 1:10 (w/
v), respectively. Thus enzyme dosage of 2% (w/v), incubation
period for 36 h and biomass:liquid ratio of 1:20 (w/v) was found
optimum for hydrolysis of algal biomass.
The effects of different pretreatments on reducing sugar yields
under optimized conditions are given in Table 2. The heat pretreatment to the biomass at a temperature of 120 C for 1 h in autoclave
with and without aqueous buffer resulted in higher reducing
sugar yields of 206.82 14.96 and 194 10.2 mg/g biomass,
respectively (p < 0.05) in comparison to the other pretreatments
investigated. The optimum reducing sugar yields obtained from
acid pretreatment with 1% H2SO4 (w/v) at 100 C for 1 h was
113.68 2.61 mg/g biomass and 178.44 9.08 mg/g when treated
with 2% liquid ammonia at a temperature of 28 1 C for 15 min
(Table 2). Both the ammonia concentration and time period had
individual signicant effects on the reducing sugar yield but their
interaction effect was found to be non-signicant by two-way
ANOVA (Supplementary Table 1). However, the heat treatment to
the biomass suspended in aqueous buffer in autoclave at 120 C
for 1 h was found optimum among all the pre-treatments studied,
and was employed for further studies.
The saccharication efciency of different commercial cellulases i.e. cellulase 22119, cellulase 22086 and cellulase 22128
(Novozyme, Denmark) under optimized pretreatment and enzymatic hydrolysis conditions for different incubation periods are described in Table 3. The highest reducing sugar yields of
215 5.04 mg/g was recorded with cellulase 22119 followed by
210 1.2 mg/g with cellulase 22086 after an incubation period of
36 h (p < 0.05). In contrast, the highest reducing sugar yield obtained from cellulase 22128 was 92.7 4.4 mg/g after an incubation period of 48 h which was remarkably inferior to the other
two enzymes investigated. The effect of different cellulase enzymes and incubation time period on reducing sugar yields was
tested by two-way ANOVA (Supplementary Table 2). The twoway ANOVA also conrmed that the choice of enzyme and the
incubation time period have remarkable signicant effects on the
enzymatic hydrolysis of algal biomass. The overall results summarized the order of reducing sugar yields as cellulase 22119
(215 5.04 mg/g) > cellulase 22086 (210 1.2 mg/g) > Viscozyme
L (206.82 14.96 mg/g) > cellulase 22128 (92.7 4.4 mg/g). The
enzyme cellulase 22119 was therefore preferred in subsequent
studies. Kim et al. (2011) also investigated the hydrolysis efciency
of different commercial cellulases on U. lactuca and showed reducing sugar yields in the range of 7.819.4% w/w while in this study
the reducing sugar yields obtained were in the range of 9.221.5%
w/w from the hydrolysis of alga U. fasciata. The higher yields of
reducing sugars realized in this study could be attributed either
to higher cellulose content in the alga investigated or to the higher
cellulolytic activity of the enzymes employed.
The reusability of the enzyme cellulase 22119 was thereafter
assessed. The reducing sugar yields obtained from the heat treatment to the biomass with and without aqueous buffer were found
to be 206.82 14.96 and 194.63 10.2, respectively (Table 2). Because of the similar yields of reducing sugars, former was selected
for rst cycle of hydrolysis and later for second cycle. The advantage offered with this strategy is that it maintains the optimum
biomass: liquid ratio. After rst cycle of hydrolysis a decline in liquid volume was observed therefore the weight of algal biomass
added in subsequent cycle was accordingly adjusted to 8 g in second and 6 g in third cycle to maintain the biomass: liquid ratio. The

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N. Trivedi et al. / Bioresource Technology 150 (2013) 106112

Table 2
The effect of different pretreatments on yields of reducing sugars from U. fasciata biomass followed by enzymatic hydrolysis.
Alga

Treatment

Conditions

Reducing sugar (mg/g)

Ulva fasciata

Acid

0.1% H2SO4 for 1 h at 100 C

0.5% H2SO4 for1 h at 100 C
1% H2SO4 for1 h at 100 C
Sodium acetate (pH 4.8), 120 C for 30 min
Sodium acetate (pH 4.8), 120 C for 60 min
Sodium acetate (pH 4.8), 120 C for 90 min
120 C for 30 min
120 C for 60 min
120 C for 90 min
1%-15 min
1%-30 min
1%-60 min
2%-15 min
2%-30 min
2%-60 min
4%-15 min
4%-30 min
4%-60 min

28.32 878c
63.36 1.18b
113.68 2.61a
180.32 8.76b
206.82 14.96a
174.82 11.32b
163.11 7.5b
194.63 10.2a
182.44 8.4a
127.66 1.76
110.03 2.04
85.7 3.06
178.44 9.08
158.42 1.82
143.43 0.59
170.04 8.92
151.07 10.83
138.33 2.33

Hot buffer

Dry heat

Liquid ammonia*

a-c

Values with different letters in columns for respective treatments were signicantly different at p 6 0.05.
The effect of liquid ammonia concentrations and treatment durations on reducing sugar yields by two way ANOVA and respective values were
signicantly different at p 6 0.05.
*

Table 3
Determination of hydrolysis potential of different commercial cellulases for hydrolyzing U. fasciata biomass.
Substrate

Ulva fasciata

Enzyme*

Cellulase 22119
Cellulase 22118
Cellulase 22086

Temperature

45 C

Reducing sugar (mg/g) after hydrolysis period

12 h

24 h

36 h

48 h

168.7 2.1
64.5 4.3
125.57 3.7

195.5 1.6
66.7 3.1
201.19 1.2

215 5.04
76.7 2.22
210.5 1.2

162.5 2.4
92.7 4.4
195.7 1.8

Values were signicantly different at p 6 0.05 as determined by two-way ANOVA.

Concentration of each enzyme used was 2% (v/v).

reducing sugar yield obtained after rst hydrolysis cycle was

2.10 0.21 g/10 g of seaweed biomass and the same increased to
3.23 0.4 g/18 g after second hydrolysis cycle. Subsequent addition of biomass (6 g) to assess the reusability of enzyme for third
time showed an abrupt decline in reducing sugar yield to
1.88 0.84 g (Supplementary Table 3). The decline in reducing sugar yield could be attributed to the adsorption of liquid over solid
biomass resulting in decline in viscosity impeding the enzymatic
hydrolysis as well as to the end product inhibition of enzyme by
glucose and cellobiose (Kuhad et al., 1999). These results showed
that the cellulase 22119 retained its activity for two cycles. Considering the fact that the biomass has 15% cellulose (Table 1), theoretical reducing sugar yield should be 1.65 g/10 g algal biomass after
rst cycle and should increase to 2.97 g/18 g after second cycle. But
both of these values were inferior to the reducing sugar yields obtained experimentally. To answer this decit, the reducing sugar
content of cellulase 22119 was determined and the same was
found to be 0.15 g/mL of the enzyme solution. The enzyme solution
(4 mL) employed for hydrolysis therefore contributed a reducing
sugar amount of 600 mg and attributes to the higher reducing sugar yield obtained experimentally. Taking into the account the
aforementioned value, the hydrolysis efciency was found to be
91% for the rst cycle and 88.5% for second cycle. Though successive saccharication by treatment of algal biomass using acid
(Khambhaty et al., 2012) and enzyme (Yanagisawa et al., 2011)
has been reported, this is the rst study wherein enzyme reusability was shown. The successive saccharication involved addition of
algal biomass and acid or enzyme in the hydrolysate obtained after
primary saccharication. On contrary, in this study algal biomass
was directly added into the primary hydrolysate without supplementing the extra enzyme while realizing the similar yields

(Supplementary Table 3) therefore present a cost effective method

to obtain higher concentrations of reducing sugars.
In addition to whole biomass, cellulose extracted from algal
biomass was also hydrolyzed under optimized conditions using
cellulase 22119. The actual reducing sugar yield obtained was
940 mg/g cellulose after subtracting the reducing sugar amount
contributed by enzyme while the same was estimated 150 mg/g
algal biomass. As the biomass was found to have 15% cellulose
(Table 1), the reducing sugar yields obtained from both these strategies were in similar range. The reducing sugar yields obtained in
the present study was superior to the reducing sugar obtained
from enzymatic hydrolysis of cellulose (870 mg sugars/g cellulose)
from G. verrucossa (Kumar et al., 2013).
3.4. Fermentation of algal hydrolysate
Fermentation efciency of the yeast strain Saccharomyces cerevisiae was evaluated on YEPD medium for different time intervals
ranging from 12 to 48 h and was considered as a control. The maximum ethanol yield of 0.51 g/g dextrose was obtained after 12 h
which showed fermentation efciency 100% considering the theoretical ethanol yield of 0.51 g/g reducing sugar (Table 4). The
hydrolysate as obtained from saccharication of 15 g dry U. fasciata
biomass contained 3.2 0.21 g reducing sugar and was used as a
substrate for fermentation by the yeast. The fermentation of this
hydrolysate for different time intervals ranging from 12 to 48 h
at 12 h incremental period gave varied ethanol yields. The rst
sampling made at 12 h fermentation period showed optimum ethanol yield of 1.28 g equivalent to 1.62 mL. Afterwards a decline in
ethanol yields of 1.17, 1.18 and 1.12 g was recorded for fermentation duration of 24, 36 and 48 h, respectively. The enzymatic

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Table 4
Laboratory scale data on saccharication and bioethanol production from U. fasciata biomass.
Biomass (g DW)

Total reducing sugar (g)

c

15
40
100
Control (YEPD)
ac

3.2 0.21
8.22 0.33b
21.82 0.19a
2.0

Fermented sugar (g)

Ethanol yield (g)

2.85 0.18
7.82 0.28b
20.70 0.25a
2.0

1.28 0.10
3.51 0.16b
9.31 0.13a
1.02

Theoretical yield

Efciency (%)

1.45
3.98
10.55
1.02

88.27
88.19
88.24
100

Values with different letters in columns were signicantly different at p 6 0.05. YEPD Yeast extract peptone dextrose medium.

Table 5
Comparison of ethanol yields reported for different macroalgal feedstocks with present study (Modied from Kumar et al., 2013).
Seaweed

Part used for hydrolysis and

fermentation

Conditions

Sugar released
(g/g)

Ethanol yield
(g/g sugar)

Efciency
(%)

References

Ulva fasciata
Gracilaria verrucosa
Kappaphycus alverzii
Gelidium amansii
Kappaphycus alverzii

Whole biomass
Pulp after agar extraction
Whole thallus
Whole biomass
Granule

Hot buffer + enzyme

Enzyme
Acidic
Dilute acid hydrolysis
Acidic

0.205
0.87 g/g cellulose
NA
0.422
0.306

0.45
0.43
0.369
0.38
0.40

88.2
84.31
72.35
74.50
80.39

Saccharina japonica
Gelidium amansii
Laminaria japonica
Sargassum fulvellum
Ulva lactuca
Gracilaria salicornia

Whole thallus
Whole biomass
Whole biomass
Whole thallus
Whole thallus
Two stage hydrolysis of fresh
biomass
Whole thallus

Thermal acid hydrolysis

Acid + enzyme
Acid + enzyme
Acid + enzyme
Acid + enzyme
Acid + enzyme

0.456
0.566
0.376
0.096
0.194
16.6

0.169
Na
0.41
NA
NA
0.079

33.13

80.39

15.49

Present study
Kumar et al. (2013)
Meinita et al. (2012)
Park et al. (2012)
Khambhaty et al.
(2012)
Jang et al. (2012)
Kim et al. (2011)
Kim et al. (2011)
Kim et al. (2011)
Kim et al. (2011)
Wang et al. (2011)

200 oC and 15 MPa for

15 min

NA

0.386

75.68

Hyeon et al. (2011)

NA

0.1330.233

26.07
45.68

Yeon et al. (2010)

Sargassum
Sagamianum
Sargassum
sagamianum

Whole biomass

Table 6
Effect of yeast extract and peptone on fermentation of algal hydrolysate.
Biomass (g DW)

Conditions

Total reducing sugar (g)

Fermented sugar (g)

Ethanol yield (g)

Theoretical yield

Efciency (%)

15
15

With yeast extract and peptone

Without yeast extract and peptone

3.2 0.21
3.3 0.12

2.85 0.18
3.0 0.28

1.28 0.10
1.35 0.15

1.45
1.53

88.27
88.23

Difference between the means were non-signicant by students t-test (p < 0.05).

hydrolysis and fermentation was then scaled up for biomass of 40

and 100 g dry weight. The respective total reducing sugars yields
on hydrolysis were found to be 8.22 0.33 g and 21.82 0.19 g
and the corresponding ethanol yields were 3.51 0.16 g and
9.31.0.13 g (Table 4).
In both these cases, the fermentation efciency was found to be
88.19 and 88.24% which is quite similar to the fermentation efciency of 88.27% recorded from the hydrolysis and fermentation
of 15 g biomass. These results clearly suggest the stability and consistency of the process described in this study.
The comparison of fermentation efciencies of reducing sugars
into ethanol reported for different macroalgal feedstock is given in
Table 5. The fermentation efciency obtained in this study was
found to be comparatively higher than those values reported earlier (Hyeon et al., 2011; Kim et al., 2011; Adam et al., 2012; Jang
et al., 2012; Khambhaty et al., 2012; Meinita et al., 2012; Park
et al., 2012; Kumar et al., 2013).
An additional fermentation experiment carried out by inoculating yeast into the fermentation broth containing algal hydrolysate
without nitrogen sources resulted into an ethanol yield of
0.450 g/g reducing sugar. The reducing sugar yields thus obtained
was equivalent to that of the same (0.449 g/g) obtained from
fermentation medium supplemented with nitrogenous sources
i.e. yeast extract and peptone (Table 6). The difference among the

means of these two experimental conditions was found to be statistically non-signicant by students t-test (p < 0.05). The seaweed
species U. fasciata investigated had protein content as high as
14.4 2.2% on dry weight basis (Table 1). Since the feedstock itself
is naturally a rich source of protein, hydrolysate as obtained following the enzymatic saccharication was fermented directly
without the addition of nitrogenous sources.
Recently, Kumar et al. (2013) and Khambhaty et al. (2012) estimated the bioethanol production at large scale as 3.8 kg/100 kg
(dry weight) from G. verrucosa and 0.23 kg/100 kg (fresh weight)
from K. alverzii. The ethanol yield estimated from the optimized
conditions in this study was 9.3 kg/100 kg of dry weight which is
equivalent to 0.93 kg/100 kg fresh weight of U. fasciata as the water
content is more than 90% of total body weight.

4. Conclusion
The present study demonstrated the utilization of the green
seaweed U. fasciata as potential marine feedstock for bioethanol
production. The ethanol yield and reducing sugar conversion efciency achieved in this study was comparatively higher than those
reported for both agarophytes and carrageenophytes. The vast
oceans around the world can gainfully be utilized for production

Author's personal copy

112

N. Trivedi et al. / Bioresource Technology 150 (2013) 106112

of marine macroalgal biomass as feedstock for chemicals and energy. Further, the seaweed farming in oceans not only helps to mitigate the global warming and climate change but also spare the
land for agriculture and forestry which are crucial for food security
of the global population.
Acknowledgements
The nancial support received from FP7 programme of European Commission under grant agreement no. 241383 is gratefully
acknowledged. Mr. Nitin Trivedi and Mr. Vishal Gupta gratefully
acknowledge the CSIR for Senior Research Fellowships. Authors
sincerely acknowledge Miss Puja Kumari, CSIR-SRF for assisting
in statistical analysis performed in this study.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2013.
09.103.
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