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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
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
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
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.
108
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
109
110
Treatment
Conditions
Ulva fasciata
Acid
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
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
111
15
40
100
Control (YEPD)
ac
3.2 0.21
8.22 0.33b
21.82 0.19a
2.0
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
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
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
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)
NA
0.386
75.68
NA
0.1330.233
26.07
45.68
Sargassum
Sagamianum
Sargassum
sagamianum
Whole biomass
Table 6
Effect of yeast extract and peptone on fermentation of algal hydrolysate.
Biomass (g DW)
Conditions
Theoretical yield
Efciency (%)
15
15
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).
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
112
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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