Algal Research 9 (2015) 4854

Contents lists available at ScienceDirect

Algal Research
journal homepage: www.elsevier.com/locate/algal

Solid state fermentation (SSF)-derived cellulase for saccharication of

the green seaweed Ulva for bioethanol production
Nitin Trivedi a,b, C.R.K. Reddy a,b,, Ricardo Radulovich c, Bhavanath Jha a,b
a
b
c

Division of Marine Biotechnology and Ecology, CSIR Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India
Academy of Scientic & Innovative Research (AcSIR), New Delhi, India
Department of Agricultural Engineering, University of Costa Rica, San Jose, Costa Rica

a r t i c l e

i n f o

Article history:
Received 22 August 2014
Received in revised form 12 February 2015
Accepted 14 February 2015
Available online xxxx
Keywords:
Bioethanol
Cellulase
Saccharication
Solid state fermentation
Ulva

a b s t r a c t
Cellulase produced from the marine fungus Cladosporium sphaerospermum through solid state fermentation
(SSF) was investigated for its saccharication potential of seaweed biomass using the common green seaweed
Ulva fasciata. The seaweed substrate, containing inoculated fungus with 60% moisture content, cultured at
25 C and pH 4 for four days, showed optimum enzyme production. The enzyme, assayed for carboxymethyl
cellulase and lter paper assay, showed an activity of 10.20 0.40 U/g and 9.60 0.64 U/g on a dry weight
basis, respectively. Further, ionic liquid tolerance of the enzyme was studied in the presence of 1-ethyl-3methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride, 1-butyl-3-methylimidazolium
triuoromethanesulfonate and 1-butyl-1-methylpyrrolidinium triuromethanesulfonate. At 10% v/v concentration,
the enzyme retained 72.17 to 85.04% activity in all the ionic liquids. The pre-incubation of enzyme in the same ionic
liquids for 24 h, the activity got slightly enhanced and ranged between 73.80 and 93.70%. The hydrolysis of U. fasciata
feedstock with enzyme (10 U/g) for 24 h at 40 C and pH 4 gave maximum yield of sugar 112 10 mg/g dry weight.
On fermentation, an ethanol yield of 0.47 g/g reducing sugar was obtained, corresponding to 93.81% conversion efciency. These ndings indicate that cellulase produced from a marine fungus can be employed for saccharication
of cellulosic feedstock for the production of renewable biofuels from marine macroalgal feedstock. Since bioethanol
yields obtained compare very favorably with those from land crops, the strategy employed in this study warrants
further exploration.
2015 Elsevier B.V. All rights reserved.

1. Introduction
According to the U.S. Department of Energy, 30% of petroleum-based
transportation fuels would be replaced with biomass-based fuels by
2025 [1]. Worldwide, the demand for renewable fuels, particularly
bioethanol, is projected to increase 3.4-fold by 2035 [2]. Cellulose, a
structural component of plant biomass, is the most abundant feedstock
used for the production of alternative liquid fuels, mainly bioethanol.
However, cellulose in terrestrial plants is intertwined with lignin, hemicelluloses and pectin, which require extra energy input as pretreatment
for their removal [3,4]. Consequently, due to their high carbohydrate content, high productivity and widespread distribution marine macroalgae
(seaweeds) are increasingly gaining prominence as an alternative renewable feedstock for sustainable production of biofuels [5]. Large scale farming of a variety of seaweeds is already practiced in a number of countries.
The current cultivated production of seaweeds worldwide is about
24 million tons (fresh weight) per annum [6]. A majority of this produce
Corresponding author at: Seaweed Biology and Cultivation Group, Division of Marine
Biotechnology and Ecology, CSIR Central Salt and Marine Chemicals Research Institute,
Bhavnagar 364002, India.
E-mail address: crk@csmcri.org (C.R.K. Reddy).

http://dx.doi.org/10.1016/j.algal.2015.02.025
2211-9264/ 2015 Elsevier B.V. All rights reserved.

is used for human consumption as food and the rest for phycocolloid extraction. Since seaweed farming is undertaken directly at sea, it does not
clash with terrestrial agricultural crops for land and fresh water resources.
Another distinct advantage of macroalgal feedstock for biofuel production
is the absence of lignin (occasionally traces), which dispenses the need for
energy-intensive pretreatment as part of the hydrolysis process prior to
fermentation. However, seaweed polysaccharides are structurally complex and diverse in chemical composition, and differ from land plants
with respect to the abundance of matrix and skeletal components. Thus,
an efcient hydrolysis for sustainable production of biofuels from different macroalgal feedstocks is required. Recently, Newman et al. [7] and
Wargacki et al. [8] genetically re-engineered microbes for efcient hydrolysis and fermentation to achieve higher yields of bioethanol from brown
seaweeds.
Earlier studies have employed either chemical or enzymatic processes for the hydrolysis of macroalgal feedstock [710]. Chemical hydrolysis (acid hydrolysis) is one of the feasible methods commonly used for
the production of fermentable sugars from lignocellulosic biomass.
There are also several studies reporting acid hydrolysis of seaweed polysaccharides for bioethanol production. The macroalgal species that have
been used include Kappaphycus alvarezii [9,10], Palmaria palmata [11],
Eucheuma cottonii [12], Undaria pinnatida [13] and Gracilaria salicornia

N. Trivedi et al. / Algal Research 9 (2015) 4854

[14]. Nevertheless, acid hydrolysis results in the production of some

non-sugar by-products such as 5-hydroxymethylfurfural (HMF), formic
acid, levulinic acid, acetic acid (organic acid), phenols and heavy metals.
These impede subsequent downstream fermentation besides causing
environmental hazards [15].
As an alternative, using enzymes obtained from microorganisms
could provide new avenues in converting complex seaweed polysaccharides into fermentable sugars. There are a few studies reporting
enzymatic saccharication of macroalgal biomass using commercial enzymes [1618]. Enzymatic hydrolysis indeed presents a green approach
but suffers from high cost of commercial hydrolysing enzymes. Since
cellulase is widely used in the production of bioethanol from cellulosic
biomass, and green seaweeds have higher cellulose content compared to both red and brown seaweeds, they are a clear choice for
saccharication.
The success of bioethanol production will greatly depend on the cost
of production of cellulase employed in the saccharication process. In
2012, it was reported [19] that cost of cellulosic ethanol production
was $0.94 per liter (40% higher than corn ethanol). More recently,
Bloomberg New Energy Finance reported that cellulosic ethanol is expected to be cost competitive with corn ethanol by 2016 [19]. It is
thus of great importance to isolate cellulase-producing microbial strains
for the efcient saccharication of plant biomass of marine origin. Cellulase production through solid state fermentation (SSF) is gaining importance over submerged fermentation. The advantages of SSF include
lower processing cost, less energy requirements, production of solid
waste, enhanced productivity, improved product stability and lower
catabolic repression [20].
To date, a majority of cellulase-producing microbes have been isolated from terrestrial sources. Little effort has been made to date exploring
marine based sources for cellulase production. Therefore, an effort was
made in this study to isolate marine microbes capable of hydrolyzing
cellulose-rich green seaweed Ulva fasciata for bioethanol production.
The present study reports the cellulase production potential of the marine fungus Cladosporium sphaerospermum through SSF and its application in saccharication of the green seaweed U. fasciata for bioethanol
production.
2. Materials and methods
2.1. Materials
All chemicals, media components and reagents used in this work
were purchased from Sigma Aldrich (U.S.A.) and Himedia laboratories
(Mumbai, India). All analyses were conducted in laboratory facilities of
CSIR-CSMCRI, Bhavnagar, Gujarat, India.
2.2. Microorganism
The cellulase-producing strain of the marine fungus
C. sphaerospermum was isolated from deteriorated seaweed Ulva cultured in the laboratory. Cellulase secretion potential was screened by
zone of clearance as qualitative measure of extracellular cellulase activity,
after ooding carboxymethyl cellulose (CMC) (1.5%) agar plates with
Lugol's iodine solution [2123]. The molecular identication of the fungal
strain was carried out by 18S rDNA sequencing using Internal Transcribed
Spacer (ITS) regions. The culture was maintained on potato dextrose agar
(PDA) at 4 C as a pure culture.

49

2.4. Solid state fermentation and optimization of parameters for cellulase

production
The green seaweed U. fasciata was used as substrate for solid state
fermentation (SSF) for cellulase production. SSF was carried out in
250-mL Erlenmeyer asks containing 10 g of dried and powdered seaweed in a modied mineral salt medium [24,25]. The mineral salt medium contained (g/L): (NH4)2SO4, 1.86; KH2PO4, 2.0; urea, 0.3; CaCl2, 0.03;
MgSO47H2O, 0.3; yeast extract, 4.08; (mg/L): FeSO47H2O, 5.0;
MnSO4H2O, 1.6; ZnSO47H2O, 1.4; CoCl2, 2.0; (w/v): peptone, 0.8%;
and, (v/v): Tween 80, 0.1%. The pH was adjusted to 5.0 before sterilization at 121 C for 15 min. Flasks were inoculated with fungal spore suspension (1 109 spores/mL) prepared in sterile 0.1% v/v Triton-X 100.
Inoculated asks were incubated at room temperature for 6 days. In
order to achieve optimum enzyme yield through SSF, cellulase production was optimized with respect to different parameters such as moisture content (40100%), incubation period (26 days), pH (26) and
incubation temperature (2540 C). After optimisation, cellulase production was also carried out using cellulose extracted from U. fasciata as a
substrate to compare enzyme production under optimized conditions.
2.5. Enzyme extraction and assay
To extract the enzyme, fermented substrate was suspended in
150 mL of 50 mM sodium acetate buffer. The broth was mixed properly
by gentle shaking before extracting cellulase. The extract was ltered
through double layered muslin cloth then centrifuged at 7000 rpm at
4 C for 15 min. The clear supernatant obtained was considered as
crude enzyme mix and used for cellulolytic assay. Total cellulase or lter
paper activity (FPase) and carboxymethyl cellulase (CMCase) activity
were determined according to Trivedi et al. [23] using lter paper and
CMC-Na as a substrate, respectively. The FPA was expressed as FPU/g
of dry substrate (FPU/g ds). One unit of enzymatic activity was dened
as the amount of enzyme that released 1 mol of reducing sugar per
minute.
2.6. Effect of pH and temperature on cellulase activity and stability
The pH and temperature optima for enzyme activity were assayed at
different pH ranging from 2 to 3 (50 mM GlycineHCl buffer); 4, 5 and 6
(50 mM Sodium acetate buffer); and at different temperatures ranging
from 4 C to 60 C with increments of 20. Further, enzyme stability was
investigated by estimating the residual enzyme activity after preincubation for 1 h at the aforementioned pH and temperature ranges.
2.7. Effect of ionic liquids on cellulase
The ionic liquids (ILs) used in this study include: 1-ethyl-3methylimidazolium acetate, [EMIM]Ac, (IL1); 1-butyl-3methylimidazolium chloride, [BMIM]Cl, (IL2); 1-butyl-3methylimidazolium triuoromethanesulfonate, [BMIM][OTF], (IL3); and,
1-butyl-1-methylpyrrolidinium triuromethanesulfonate, [BMPL][OTF],
(IL4). Enzyme activity was determined at different IL concentrations
(1%, 5%, 10%, 15% and 20% v/v) under optimized assay conditions and
was compared with the control reaction carried out in aqueous medium.
Further, the IL stability of the enzyme was investigated by estimating enzyme activity after pre-incubation for different time intervals ranging
from 24 to 96 h in different ILs (10% v/v). The ILs did not affect pH values
in the enzyme assay mix which remained at pH around 5.0.

2.3. Collection of algal sample

2.8. Hydrolysis of algal biomass through SSF-derived cellulase
The marine macrophytic green alga U. fasciata Delile was collected
from Veraval (N 20 54.87, E 70 20.83), coast of Gujarat, India. Seaweed samples were washed thoroughly with tap water to remove
salts, epiphytes and debris and dried to a constant weight at 50 C.
After drying, samples were powdered using a grinder.

Enzymatic hydrolysis of green seaweed U. fasciata mass was optimized with SSF-derived cellulase with respect to enzyme dosage, hydrolysis period, temperature and pH. For this, dried algal powder (1 g)
was hydrolysed with different enzyme dosages (5, 10 and 15 U/g

50

N. Trivedi et al. / Algal Research 9 (2015) 4854

biomass) in a xed volume (25 mL) of sodium acetate buffer (pH 4.0
0.5). This was incubated for different time intervals from 12 to 48 h at
40 C on an orbital shaker with a speed of 150 rpm. Samples were
taken out periodically after an interval of 12 h each and centrifuged.
The reducing sugar was measured spectrophotometrically using the 3,
5-dinitrosalisylic acid (DNS) method [26].
After optimization of enzyme dosage and incubation period, hydrolysis temperature (30 C to 50 C) and effect of pH (26) on biomass hydrolysis were also optimized under standard assay conditions. The algal
biomass hydrolysis with SSF-derived cellulase (extracted using algal
biomass and cellulose as substrate) was qualitatively analyzed using
thin layer chromatography (TLC) by spotting hydrolysate sample on
LK 60 Whatman silica gel plates. The solvent system used for separation
was a mixture of butanol, ethanol and water at a ratio of 5:5:3. For sugar
detection, the silica gel plate was sprayed with 5% sulfuric acid in methanol followed by heating at 100 C for 15 min. Glucose was used as a
standard at a concentration of 20 mg/mL [25]. The conrmation of
biomass hydrolysis was also carried out using gel permeation chromatography (GPC) (Water Alliance, model 2695) equipped with GPC
column ultra hydrogel 120 and 500 and refractive index detector (Waters 2414).

Table 1
Optimization of different SSF parameters for maximizing enzyme production.
Parameters

Conditions

Enzyme activity in U/g of dried seaweed

for different substrates and conditions
CMC (CMCase)

Moisture

pH

Incubation period

Temperature

40%
60%
80%
100%
2
3
4
5
6
2 days
4 days
6 days
25 C
30 C
35 C
40 C

9.14 0.50
11.04 0.64a
7.52 0.43c
6.00 0.30d
5.41 0.30e
6.81 0.60d
10.20 0.40a
9.10 0.50b
7.60 0.40c
6.50 0.60b
9.60 0.53a
5.10 0.30c
10.80 0.60a
6.10 0.70bc
5.60 0.42cd
5.00 0.43d

Filter paper (FPase)

7.90 0.35bc
9.20 0.20a
7.60 0.30c
6.91 0.32d
4.41 0.40e
5.61 0.34d
9.60 0.64a
7.60 0.60b
6.34 0.54c
4.90 0.30c
8.83 0.40a
6.14 0.74b
9.00 0.32a
4.94 0.31b
4.00 1.14c
3.91 0.31c

Values followed by the same letter are not signicantly different at p b 0.05.

Among the different microbes isolated from deteriorating Ulva, the

fungal strain showed maximum zone of clearance on CMC agar plates
after ooding with Lugol's iodine and was, thus, selected for further
studies. The fungal strain was identied as C. sphaerospermum. The
DNA sequence for the isolated strain was submitted to NCBI GenBank
with Accession No. KJ546144.

enzyme activity increased with increasing moisture content up to 60%

and thereafter declined. Enzyme activity for 60% moisture content was
the highest at 11.04 0.64 U/g for CMCase and 9.2 0.20 U/g for
FPase, while it was the lowest for 100% moisture content, at 6.0
0.30 U/g and 6.91 0.32 U/g, respectively (p b 0.05). Maurya et al.
[28], while studying cellulase production from Trichoderma reesei, also
reported similar trends in enzyme activity for different moisture levels
(4090%). The higher enzyme production with 60% moisture content
of substrate in the present study is in agreement with previous ndings
[29,30].
For incubation period, CMCase and FPase activities were the highest
for day four, at 9.60 0.53 U/g and 8.83 0.40 U/g, respectively, while
respective values declined to 5.10 0.30 U/g and 6.14 0.74 U/g for
day six (Table 1). These results are in accordance with cellulase produced through SSF from Aspergillus niger NS-2 [29], where optimum enzyme production was on day four, with activity of 2.830.6 U/g.
Effects of substrate pH (26) and temperature on enzyme production were also signicant (Table 1). The acidic range of substrate
(pH 46) signicantly favored optimal enzyme production (p b 0.05).
Optimum enzyme production was observed at pH 4 with an activity of
10.2 0.40 U/g for CMCase and 9.60 0.64 U/g for FPase. Similar
results were reported for cellulase produced from T. reesei [28] and
Aspergillus terreus [25]. Enzyme production was highest at 25 C,
10.80 0.60 U/g for CMCase and 9.0 0.32 U/g for FPase, whereas at
40 C values declined to 5.0 0.43 and 3.91 0.31 U/g, respectively
(p b 0.05). In another experiment, cellulase produced under optimized
SSF conditions using as substrate cellulose extracted from U. fasciata,
also showed enzyme activity of 10.51 0.61 U/g for CMCase and
8.11 1.0 U/g for FPase, corroborating those obtained with Ulva
feedstock.

3.2. Optimization of SSF for enzyme production

3.3. Effect of pH and temperature on cellulase activity and stability

Data on enzyme production and optimization of SSF are summarized

in Table 1. The study showed that enzyme activity signicantly varied
with variations in moisture content of substrate, pH, temperature and
incubation period (p b 0.005) (Table 1). The dry algal substrate containing inoculated fungus with 60% moisture content, incubated for 4 days
at 25 C and pH 4, showed optimum enzyme production. The enzyme
analyzed for CMCase showed an activity of 10.2 0.40 U/g and for
FPase of 9.6 0.64 U/g, although marginal variations in activity were
observed between batches. The substrate with high moisture content
prevents oxygen penetration whereas low moisture content leads to
the inhibition of microbial growth owing to poor accessibility to nutrients, causing decreased enzyme activity [27]. In the present study,

Enzyme activity, found to be optimal at pH 4 and 40 C, respectively,

showed linear increase with increasing pH from 2 to 4 and thereafter
decreased to 24% and 45% at pH 5 and 6, respectively. The enzyme
was also found to be stable over a broad range of pH 24 while at
pH 5 and 6 the enzyme activity was 61% and 33%, respectively. Although
the enzyme showed activity over a broad range of temperatures (4 to
60 C), it was highest at 40 C. At higher temperature (60 C), enzyme
activity signicantly declined to 19% from that of optimum activity.
Thermal stability analysis revealed an increase in enzyme activity with
increasing temperature. Activity at 4 C was 68.30% while at 40 C it
showed a substantial increase to 90.70%, declining slightly to 85.8% at
60 C (data not shown).

2.9. Fermentation of algal hydrolysate

Fermentation of algal hydrolysate was carried out using Saccharomyces cerevisiae MTCC No. 180 according to a method reported by Trivedi
et al. [18]. The algal hydrolysate obtained after enzymatic hydrolysis of
10 g of biomass was enriched with peptone (5 g/L) and yeast extract
(3 g/L). The broth was inoculated with fresh yeast culture and incubated
for 12 h at 28 2 C on an orbital shaker with a shaking speed of
120 rpm. Samples were withdrawn after 12 h and analyzed for ethanol
yield and residual reducing sugars by gas chromatographymass spectrometry (GCMS) and DNS method, respectively.
2.10. Statistical analysis
All the experiments were conducted with three replicates and results are presented as mean standard deviation (S.D.). Differences between mean values were evaluated by one way analysis of variance
(ANOVA).
3. Results and discussion
3.1. Microorganism

N. Trivedi et al. / Algal Research 9 (2015) 4854

3.4. Effect of ILs on cellulase enzyme

Both activity and stability of the enzyme varied across the four IL
types and their concentrations investigated in this study. At 5% v/v concentrations, enzyme activity was found to be highest in IL1 (89.60%)
followed by IL3 (84.94%), IL4 (82.52%) and IL2 (73.30%) (Fig. 1a). The
enzyme retained its activity in all ILs at 10% v/v, ranging from 72.20%
to 85.04%. At the highest IL concentration tested (20% v/v), enzyme activity ranged from 51.82% (IL2) to 70.60% (IL1). The residual activity of
70.60% obtained in the presence of 20% (v/v) [EMIM]Ac, which is the
gold standard for biomass treatment [23], was superior than those
reported from fungal strain T. reesei (55%) and Thermotoga maritime
(40%) [31,32], and lower than those reported from fungal strain
Pyrococcus horikoshii (95%) [32] and from a bacterial strain of
Pseudoalteromonas sp. (94.40%) [23]. In our previous study [23]
with bacterial cellulase, the enzyme activity in all ILs at 20% (v/v)
concentration was in the order of [EMIM]Ac (94.40%) N [BMPL][OTF]
(80.20%) N [BMIM][OTF] (74.70%) N [BMIM]Cl (73.20%) whereas in the
present study enzyme activity was found to be lower yet in the same
order of ILs with 70.60% N 69.10% N 58.10% N 51.82%, respectively.
The IL stability (10% v/v) of the enzyme was also studied for different
pre-incubation intervals from 24 to 96 h under the optimized assay
conditions (Fig. 1b). The order of enzyme activity after 24 h of preincubation was IL1 (93.70%) N IL2 (84.20%) N IL4 (74.10%) N IL3
(73.80%). Enzyme activity declined after increasing the pre-incubation
period from 48 to 96 h with IL1 and IL2. Interestingly, enzyme activity

51

increased with IL3 and IL4 after pre-incubation period from 48 to

72 h; thereafter, it declined after 96 h in the order of IL1 (75.20%)
followed by IL4 (64.33%), IL2 (61.52%) and IL3 (56.54%).
3.5. Hydrolysis of algal biomass through SSF-derived cellulase
The saccharication of U. fasciata mass with SSF-derived cellulase
was optimized with respect to enzyme dosage, hydrolysis period, temperature and pH. Different enzyme dosages of 5, 10 and 15 U/g yielded
signicantly different (p b 0.05) reducing sugar contents of 48.70
4.30, 118.22 3.20 and 98.22 3.50 mg/g biomass, respectively
(Fig. 2a). The enzyme dosage of 10 U/g was found to be optimum and
was, thus, subsequently employed for hydrolysis of biomass. The optimization of enzymatic hydrolysis period is shown in Fig. 2b. Reducing
sugar yields were found to increase linearly with increasing incubation
period from 12 to 24 h, ranging between 82.20 6.0 and 114.20
1.50 mg/g, whereas those of 3648 h showed marginally increased
values between 117.42 4.84 and 123.92 3.0 mg/g (p b 0.05). Further increase in incubation period to 60 h showed a decline in reducing
sugar yield to 110.82 4.93 mg/g. Since the difference in reducing
sugar yields between 24 and 48 h, though signicant, was b 10%, 24 h
of incubation period was considered here as optimum for subsequent
studies.
With increase in pH from 2 to 4, reducing sugar yields were also increased signicantly and ranged between 80.30 2.52 and 115.52
1.60 mg/g (p b 0.05). At higher pH 5 and 6, reducing sugar yields declined to 92.10 3.03 and 74.21 2.84 mg/g, respectively (Fig. 2c).
The optimization of biomass hydrolysis temperature allowed to obtain
the optimum reducing sugar yield of 113 2.63 mg/g at 40 C, whereas
at 30 C and 50 C yields were 100 4.34 and 107.19 2.70 mg/g, respectively (p b 0.05) (Fig. 2d). Thus, enzyme dosage of 10 U/g, incubation period of 24 h, hydrolysis temperature of 40 C and pH 4 were
found to be optimal for the hydrolysis of algal biomass. To further conrm
the reproducibility of these ndings, hydrolysis was carried out using 10 g
batch of algal biomass under optimized conditions which gave a reducing
sugar yield of 1.12 0.1 g corresponding to 11.2% (w/w) on dry biomass.
Recently, Trivedi et al. [18] reported reducing sugar yields of 9.2 to
21.50% (w/w dry biomass) from the hydrolysis of U. fasciata biomass
using commercially sourced cellulase. Similarly, Kim et al. [16] also reported higher yield of reducing sugars (7.8 to 19.4% w/w dry biomass)
from saccharication of Ulva lactuca using commercial cellulase. Though
the optimized reducing sugar yield of the present study was lower than
earlier reported studies, it showed the potential of SSF-derived cellulase
for the hydrolysis of the green seaweed U. fasciata biomass. This is the
rst study where SSF-derived cellulase was used for the hydrolysis of
seaweed biomass.
The resulting hydrolysate was further subjected to fermentation to
produce bioethanol. Prior to this, the presence of glucose in the hydrolysate, determined with TLC, conrmed the hydrolysis of cellulose into
glucose (Fig. 3). Further, the GPC spectrum for enzymatically hydrolysed products also conrmed the degradation of polymers into low
molecular weight fractions with a molecular weight of 159 g/mol,
evidencing the formation of monosaccharides. Therefore, the formation
of monomeric units (glucose) in the hydrolysate indirectly provided evidence for the occurrence of functionally-active cellulase mixture of all
the three types in the crude enzyme mix.
3.6. Fermentation of algal hydrolysate

Fig. 1. Effect of different ionic liquids on cellulase activity (a) and stability (b) at 10% IL (v/v)
concentration.

The hydrolysate obtained from the saccharication of 10 g dry

U. fasciata biomass, containing 1.12 0.1 g reducing sugar, was used
as a substrate for fermentation by the yeast strain S. cerevisiae MTCC
No. 180. The optimum ethanol yield was found to be 0.44 0.10 g
with 93.81% conversion efciency after 12 h of fermentation (Table 2).
The ethanol yield obtained in this study was found to be superior [8,

52

N. Trivedi et al. / Algal Research 9 (2015) 4854

Fig. 2. Effect of enzyme dosage (a), incubation period (b), pH (c) and temperature (d) on the enzymatic hydrolysis of U. fasciata feedstock. Values followed by the same letters are not
signicantly different at p b 0.05.

10,16,33-37] and comparable [38,39] to earlier reported studies

(Table 3).
Recently, Yoza and Masutani [40] and Trivedi et al. [18] reported
bioethanol production from Ulva reticulate and U. fasciata, respectively,
using commercial hydrolytic enzymes whereas ethanol production in

the present study used SSF-derived cellulase enzyme. The ethanol

yield (0.44 kg/100 kg FW) obtained here is higher than values reported
from the red seaweed Gracilaria verrucosa (0.38 kg/100 kg FW) [34].
Bruhn et al. reported an estimated production of 45 tons DW/ha/y of
Ulva on land based cultivation experiments, which were found to be
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 [41].
Assuming yields of 45 tons DW/ha/y from sea farming, results obtained here represent an initial capacity to produce bioethanol of
2509 L/ha/y, which is superior to bioethanol yields produced from
wheat, sorghum, maize and paddy rice and inferior to those from poplar
and miscanthus [4244]. Therefore, the green seaweed Ulva holds substantial potential as an alternative feedstock for the production of
bioethanol.

4. Conclusions
This is the rst report where SSF-derived cellulase has been used for
the saccharication of cellulosic-rich green seaweed biomass. The
hydrolysis of U. fasciata feedstock with SSF-derived cellulase under optimized conditions yielded reducing sugars of 112 10 mg/g DW which
on fermentation gave an ethanol yield of 0.47 g/g reducing sugar, accounting for 93.81% conversion efciency. The strategy employed in
this study represents a greener approach for the saccharication of
cellulosic-rich algal biomass and thus its use for the production of
algal bioethanol at the commercial scale warrants further exploration.

Table 2
Laboratory scale data on saccharication and bioethanol production from U. fasciata
feedstock.

Fig. 3. TLC analysis of algal hydrolysate obtained following the saccharication with cellulase produced from algal cellulose (a) and algal biomass (b) as feedstock. Standard glucose
(c) was used as control.

Biomass
(g DW)

Total reducing
sugar (g)

Fermented
sugar (g)

Ethanol
yield (g)

Theoretical
yield (g)

Efciency
(%)

10

1.12 0.10

0.92 0.13

0.44 0.10

0.47

93.81

N. Trivedi et al. / Algal Research 9 (2015) 4854

53

Table 3
Comparison of ethanol yields reported for different macroalgal feedstocks.
Macroalgae

Mode of hydrolysis

Fermenting strain

Ethanol yield (g/g reducing sugar)

References

Ulva fasciata
Eucheuma cottonii
Gracilaria sp.
Gracilaria verrucosa
Eucheuma cottonii
Kappaphycus alvarezii
Gelidium amansii
Sargassum sagamianum
Sargassum sagamianum
Laminaria japonica
Saccharina japonica

Enzymatic
Acid
Sequential acid and enzymatic hydrolysis
Enzymatic
Amberlyst TM-15 (Catalyst)
Acid
Dilute acid hydrolysis
Thermal liquefaction
Thermal liquefaction
Acid + enzymatic
Engineered microbial enzyme

Saccharomyces cerevisiae MTCC No. 180

Saccharomyces cerevisiae
Saccharomyces cerevisiae Wu-Y2
Saccharomyces cerevisiae HAU
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Brettanomyces custersii KCTC 18154P
Pichia stipitis
Pichia stipitis CBS 7126
Ethanologenic strain E. coli KO11
Engineered BAL1611

0.47
0.39
0.48
0.43
0.33
0.37
0.38
0.39
0.430.44
0.40
0.41

Present study
[35]
[39]
[34]
[36]
[10]
[33]
[37]
[38]
[16]
[8]

Acknowledgments
The partial nancial support received from BioWALK4Biofuels from
the European Commission under grant agreement no. 241383 is gratefully acknowledged. Mr. Nitin Trivedi gratefully acknowledges the
CSIR, New Delhi for a Senior Research Fellowship (SRF).
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