Accepted Manuscript

Bioethanol production from the macroalgae Sargassum spp

Myra G. Borines, Rizalinda I. de Leon, Joel L. Cuello

PII: S0960-8524(13)00476-8
DOI: http://dx.doi.org/10.1016/j.biortech.2013.03.108
Reference: BITE 11568

To appear in: Bioresource Technology

Received Date: 2 December 2012

Revised Date: 15 March 2013
Accepted Date: 17 March 2013

Please cite this article as: Borines, M.G., de Leon, R.I., Cuello, J.L., Bioethanol production from the macroalgae
Sargassum spp, Bioresource Technology (2013), doi: http://dx.doi.org/10.1016/j.biortech.2013.03.108

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 1

Bioethanol production from the macroalgae Sargassum spp.

Myra G. Borinesa*, Rizalinda I. de Leonb, Joel L. Cuelloc

a,
Department of Chemical Engineering , College of Engineering and Agro-industrial

Technology, University of the Philippines Los Baos, College,4031, Laguna, Philippines

(E-mail: mgborines@yahoo.com)
b
Department of Chemical Engineering, College of Engineering, University of the

Philippines Diliman, 1101, Quezon City, Philippines (E-mail: rldeleon@up.edu.ph)

c
Department of Agricultural and Biosystems Engineering, University of Arizona, Tucson,

Arizona, 85721, USA (E-mail: cuelloj@email.arizona.edu)

Abstract

Macroalgae, an abundant and carbon-neutral renewable resource, with several species rich

in carbohydrates are suitable for bioethanol production. This study focused on the

pretreatment, enzyme saccharification and fermentation of Sargassum spp., a brown

macroalgae for bioethanol production. The optimal acid pretreatment condition achieved in

terms of glucose and reducing sugar yields was 3.4 to 4.6 % (w/v) H2SO4 concentration,

115 0C and 1.50 h. The pretreated biomass was hydrolyzed with cellulase enzyme system

supplemented with -glucosidase. After fermentation by Saccharomyces cerevisiae at 40

0
C, pH of 4.5 for 48 h, the ethanol conversion rate of the enzyme hydrolysate reached 89%,

which was markedly higher than the theoretical yield of 51% based on glucose as

substrate. Since all the glucose was consumed during fermentation, other sugar sources

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might be present in the hydrolysate . The macroalgae, Sargassum spp., showed significant

potential as a renewable feedstock for the production of bioethanol.

Keywords: bioethanol; Sargassum; macroalgae; fermentation; alternative fuel

1.0 Introduction

Globally, fossil-fuel dependence resulted in different environmental problems, which

include global warming, air quality deterioration, oil spills and acid rain among others. The

combustion of fuel emits carbon dioxide (CO2), one of the most significant greenhouse

gases that trap heat in the earths atmosphere. The level of atmospheric CO2 concentration

was reported to be around 350-380 ppm in 2010 and is predicted to increase to 450 ppm by

2020 if no action is taken (Kraan, 2010).

The heightened awareness of global warming as well as other environmental issues has

increased interest in the development of methods to mitigate greenhouse gas (GHG)

emissions and lessen the production of pollutants. One of these abatement methods is the

use of biofuels made from renewable sources of energy (Jang et al., 2012; Meinita et al.,

2011).

The use of biofuel-blended gasoline and diesel has grown in popularity in several countries

not only to bring environmental benefits but as well as to establish cost-competitive

domestic energy resources and to generate additional economic development. One of the

alternative non-petroleum-based sources of energy being considered worldwide is

bioethanol. Majority of the bioethanol produced worldwide comes from terrestrial

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biomass which is essentially food (Kraan, 2010; John et al., 2011). However, food crop-

based bioethanol production has brought issues on food security and the usage of

pesticides, arable land and fresh water in their cultivation (Khambhaty et al, 2012; Kraan,

2010). Compounding problems associated with the bioethanol production from these

sources have led to the development of a more sustainable alternative feedstock.

Lignocellulosic biomass such as wood, agricultural, or forest residues has the potential to

be an alternative bioenergy source. But due to its structural complexity, some form of

pretreatment is necessary to make biomass more susceptible to enzymatic attack (Wi, et al.,

2009). The current pretreatment methods employed on lignocellulosic materials are

relatively costly and low yield (Balat et al., 2008; John et al., 2011). Although

lignocellulosic biofuels do not compete with food as a feedstock, they do however;

compete for land and fresh water resources (Kraan, 2010).

Macroalgae, an abundant and carbon-neutral renewable resource, are now considered as a

third-generation biomass that can be used in bioenergy production (Jang et al., 2012). This

biomass, with several species rich in carbohydrates and is known to contain low lignin or

no lignin at all, is suitable for bioethanol production (Yanagisawa et al., 2011). The

production of this crop is also believed to be sustainable. The use of non-food macroalgae

for bioethanol production has other advantages such as reduced competition with

agricultural food and feed crops, high yields per area and non-dependence on agricultural

fertilizer, pesticides, farmable land, and freshwater (Adams, 2009; Kraan, 2010).

Furthermore, macroalgal growth consumes large amounts of CO2 and there are pre-existing

markets for bioethanol macroalgae wastes (Borines et al., 2011).

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Although macroalgae are rich in carbohydrates and many of these species are

commercially important as phycocolloids, only few studies dealt on its saccharification and

fermentation (Khambhaty et al., 2012). In general, bioethanol production from biomass

involves pretreatment, enzymatic hydrolysis, fermentation and distillation. The

saccharification of macroalgae is important prior to ethanol fermentation. Various physical,

chemical and biological pretreatments have been studied to increase the saccharification

efficiency.

Recently, macroalgae are gaining wide attention as an alternative renewable source of

biomass for the production of bioethanol. Various researches have been conducted on the

utilization of the marine alga biomass as bioethanol feedstock. Different macroalgae

groups such as Laminaria (Adams et al., 2009; Ge et al., 2011; Horn et al., 2000, & Kim et

al. 2011) , Sacchoriza and Alaria (Yeon et al. 2011), belonging to brown seaweed, and red

algal species such as Gelidium amansii (Yoon et al. 2010), Kappaphycus alvarezii (

Khambhaty et al., 2012; Meinita et al., 2011), and Gracilaria salicornia (Wang et. al,

2011) have been considered as potential sources for bioconversion to ethanol. Isa et al.

(2009) utilized the green macroalgae, Ulva spp. as bioethanol feedstock. The feasibility of

producing bioethanol from brown seaweed, Sargassum sagamianum, was also investigated

by Yeon et al. (2011).

As the interests in macroalgae for bioethanol production are growing, intense research is

required for the efficient utilization of this biomass. Currently, the use of macroalgae to

produce fuel ethanol faces significant technical and economic challenges and its success

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depends on the development of an environmentally-friendly pretreatment methods, highly

effective enzyme systems and efficient microorganisms to convert fermentable sugars to

ethanol.

Sargassum, a brown macroalga, is widely distributed in tropical and subtropical seas. It is

the most dominant and abundant alginophyte in tropical areas. In the Philippines, the

Sargassum species are the largest among the marine algae but is still underdeveloped and

minimally utilized among locally available seaweeds. (Trono & Ganzon-Fortes, 1988).

This study focused on the dilute acid pretreatment, enzyme saccharification and

fermentation of Sargassum spp. The optimal pretreatment conditions were identified and

the ethanol conversion rates were measured at different fermentation conditions.

2.0 Materials and methods

2.1 Biomass

Sargassum spp. were harvested in the coastal waters of Bolinao, Pangasinan, Philippines.

Samples were washed with tap water, drained, cut and air dried. The samples were then

oven dried at 70oC and was ground in a Hammer mill to pass through a 35 Tyler mesh

sieve. The samples were stored in a clean air-tight container and used for subsequent

experiments.

Proximate analysis of the biomass consists of the determination of the samples moisture

or volatile matter, crude ash, crude fat, crude protein, crude fiber and carbohydrates was

done based on the method of Martinez-Goss, et al. (2001). The moisture content of the

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dried samples was determined by heating the sample in an oven maintained at 105 0C for at

least 5 h or until it reached a constant weight. The sample from the moisture determination

was ignited in a muffle furnace at 550 0C for 2 h or until a stable weight was reached.

Crude fat was determined using the Soxhlet method by extraction using petroleum ether as

solvent. The sample was placed in the Soxhlet flask and extracted with ether for 16 h. The

solvent was then recovered and the flask containing the fat was dried, cooled and weighed.

The crude fiber content of the sample was determined by Weende Method where fiber is

separated from other components of the sample by boiling with sulfuric acid to hydrolyze

the carbohydrates, protein materials and some minerals. The crude protein was measured

using Kjeldahl method using a general factor of 6.25 for algae. The carbohydrate content

of the sample was calculated by subtracting the sum of moisture, ash, crude fat, crude fiber

and crude protein from 100. The holocellulose and alpha-cellulose analyses were done

based on the methods of Erickson (1962) and Teramoto (2008), respectively.

The mannitol of the biomass was extracted using the method proposed by Percival and

McDowell (1967). For every gram of finely ground seaweed, 10 ml of water at pH 2.0

(adjusted using hydrochloric acid, HCl) was used to extract the mannitol and other water

soluble at 65 0C for 1 hour. Extracts were initially filtered using fine cheesecloth and

finally using a glass fiber filter. Extracts were analyzed for mannitol and other

monosaccharide present using High performance liquid chromatography (HPLC) and total

reducing sugar using the DNS (dinitrosalicylic acid) method (Miller, 1959).

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2.2 Optimization of the dilute acid hydrolysis of algal biomass

Preliminary runs were conducted using the two level factorial design to evaluate the effects

of hydrolysis variables: sulfuric acid concentration, temperature and reaction time on

glucose and reducing sugar yields after enzyme saccharification. Since the two-factorial

design cannot provide sufficient information to adequately model the true surface of the

response, especially the curvature and to eventually pinpoint the optimal pretreatment

conditions, it was necessary to create additional experimental points using the central

composite design (CCD). CCD was used in the optimization study using triplicate runs for

each combination: sulfuric acid concentrations (1.0 - 5.0 w/v); temperatures (115 - 126 0C)

and reaction time (0.5 - 1.5 h). The factorial points were augmented with center points and

axial points. The experimental design matrix is shown in Table 1. Control treatment was

also done in triplicate runs. One (1) g of ground biomass was mixed with sulfuric acid to

final concentrations of 0.0% to 6.36% (w/w) at solid loading of 10% (w/w) and incubated

at different temperatures (111 - 130 0C) with residence time of 0.16 to 1.84 h. Pretreated

samples were thoroughly washed with approximately 300 ml of hot water, centrifuged and

decanted to separate the acid treated biomass. Biomass was then washed to remove the

inhibitory materials produced during pretreatment which might affect the enzymatic

hydrolysis and fermentation (McMillan, 1994).

The acid treated biomass was then subjected to enzyme saccharification. About 10 ml of

0.1 M citrate buffer at pH 4.8 was added to each sample and the volume was completed to

20 ml by the addition of deionized water. The pH was adjusted to 4.8 using NaOH. To

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prevent the growth of organisms during the digestion, 200 L of 2% sodium azide was

added to the sample (Selig, et al., 2008). The samples were then brought to a temperature

of 50 0C by warming in the water bath shaker set at 50 10C. After equilibration, the

appropriate volumes of cellulase enzyme preparation and -glucosidase (cellobiase)

enzyme were added to each vial at the same time. The samples were then placed back in

the water bath shaker and incubated at 100 rpm for a period of 48 h. The samples were

hydrolysed using 50 FPU cellulase/ g biomass and 250 CBU cellobiase/ g biomass. The

cellulase from Trichoderma reeseii ATCC 26921 and cellobiase (-glucosidase) from

Aspergillus niger (Novozyme 188) were purchased from Sigma-Aldrich Co. After

hydrolysis was completed, the residues were separated from liquid by centrifugation,

decantation and filtration. The sugar in the liquid was then analyzed.

2.3 Effect of enzyme loading and hydrolysis time on enzyme saccharification of the

acid-pretreated biomass

Three (3) g of ground biomass was mixed with 4 % (w/v) sulfuric at solid loading of 10%

(w/w) and incubated at 115 0C for 1.5 h. Pretreated samples were thoroughly washed with

approximately 400 ml of hot water, centrifuged and decanted to separate the acid treated

biomass.

The acid treated biomass was then subjected to enzyme saccharification. About 30 ml of

0.1 M citrate buffer at pH 4.8 was added to each sample and the volume was completed to

60 ml by the addition of deionized water. The pH was adjusted to 4.8 using NaOH and 600

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L of 2% sodium azide was added to the samples. The samples were then brought to a

temperature of 50 0C by warming in the water bath shaker set at 50 10C. To each vial, the

appropriate volumes of cellulase enzyme preparation and -glucosidase(cellobiase)

enzyme were added accordingly at the same time. The samples were then placed in the

water bath shaker set 50 10C and 100 rpm. The samples were hydrolysed using 5 and 50

FPU cellulase/ g biomass and 0 and 250 CBU cellobiase/ g biomass. The course of the

enzyme hydrolysis was monitored for the acid-treated biomass subjected at different

enzyme loadings by monitoring the reducing sugar and glucose released for a period of 0,

8, 24, 48, 72 and 96 h.

2.4 Fermentation of algal hydrolysate

After the dilute acid hydrolysis with 4% H2SO4 (w/v) at 115 0C for 1.5 h, the seaweed

slurry was washed with hot water centrifuged and decanted to separate the acid treated

biomass. The biomass was then subjected to enzyme saccharification as in the previous

method using an enzyme loading of 10 FPU cellulase/ g biomass and 250 CBU cellobiase/

g biomass. The samples after hydrolysis were centrifuged, decanted and filtered to separate

the liquid hydrolysate from the residue. The pH of the samples was adjusted accordingly
0
using NaOH. The enzymatic hydrolysate was sterilized at 120 C for 20 min.

Supplementary nutrients were added to the fermentation medium at a final concentrations

of 0.9 g/Li (NH4)2SO4 and 0.375 g/Li yeast extract (Ge at al., 2011). The medium was

inoculated with 0.5 g/Li of dried Saccharomyces cerevisiae obtained from Pinal Energy,

Pinal County, Arizona. The flasks were sealed with rubber stoppers through which

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hypodermic needles had been inserted for the removal of CO2 produced. The fermentation

was performed in flasks at pH 4.5 and 6.5, incubation temperatures of 30 and 40 0C and

fermentation time of 48 and 72 h. Samples were withdrawn after fermentation and

analysed for glucose, reducing sugar and ethanol content. Fermentation experiments were

performed in triplicate.

2.5 Chemical Analysis

The total reducing sugar of the samples was determined by the DNS (dinitrosalicylic acid)

method using glucose as standard (Miller, 1959). The color formed was measured at 550

nm using the Ocean Optics optical fiber spectrophotometer, SpectraSuite USB 4000.

Glucose, mannose, cellobiose and mannitol in the enzyme hydrolysates were analysed by

High performance liquid chromatography (HPLC). The samples were filtered through 0.20

m nylon 30-mm membrane syringe filter prior to analysis. The HPLC analysis was

conducted using a Shimadzu LC-9A Liquid Chromatograph equipped with Shimadzu

Column Oven CTO-6A, a Shimadzu Autoinjector SIL 6-A, a Shimadzu Refractive index

detector, RID-6A, using a 300 x 7.8 mm Rezex-RPM Monosaccharide Pb 2+ Column and

2+
4 x 3 mm Microguard Pb column. The analysis was done with an oven temperature of

80 0C and detector temperature of 40 0C using HPLC water as mobile phase at a flow rate

of 0.50 mL/min. The amount of sugars was quantified by comparing peaks with those of

standard sugars of known concentrations.

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The ethanol and sugar in the fermentation broth were analysed using the Shimadzu LC-

20A Liquid Chromatograph and 300 x 7.8 mm Rezex-ROA Organic Column (Phenomenex

Inc., Torrance, CA, USA). The analysis was done at 32 0C using 2.5 mM H2SO4 as eluent

at a flow rate of 0.5 mL/min. The amounts of ethanol and sugar were quantified by

comparing peaks with those of standard solutions of the analytes with known

concentrations.

2.6 Scanning Electron Microscopy

The untreated and treated samples were mounted in a conductive tape and coated

with Pt prior to scanning electron microscopy analysis. The Field Emission Scanning

Electron Microscope (Hitachi S-4800 Type II Ultra High Resolution) equipped with

Energy Dispersive X-ray Spectroscopy detector was used to obtain the micrograph,

elemental maps and spectrum of the samples.

2.7 Statistical Analysis

The experimental data were statistically analysed using Design Expert 8.01 (Stat-Ease,

Inc.) software. In addition to T-test, the effects of the pretreatment variables were also

subjected to T-test with Bonferroni correction to make multiple pairwise comparisons

between groups of samples. A given alpha value () may be appropriate for each

individual comparison but it may not be applicable for the set of all comparisons. So to

avoid a lot of false positives, the alpha value needs to be lowered to account for the number

of comparisons being performed. For the entire set of n comparisons, the Bonferroni

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correction sets that the alpha value equal to /n. The effects that are above the Bonferroni

limit are almost certainly significant and should be added to the model while the effects

that are above the t-value limit are possibly significant and should be added if they make

sense to the experimenter. However, effects that are below the t-value limit are not likely

to be significant.

The optimal conditions were determined using the response surface method based on the

predictive models generated.

3.0 Results and discussion

3.1 Compositional analysis of biomass

The EDS spectrum of the dried sample showed that the major elemental components of

the sample are carbon and oxygen, 56.81% and 30.58 % by weight, respectively. Other

elements such as Na, Mg, S, Zr, Ca, K, and Cl were also present in the sample but only in

small amount, less than 1% by weight.

The proximate analysis of the dried sample is shown in Table2. On a dry basis, the amount

of carbohydrate in the sample was 47.06 %, which is within the reported per cent

carbohydrate content of Sargassum species. The ash content of the sample on dry basis,

29.48%, is lower compared to S. fulvellum, 46% (Kim et al., 2011) and S. polycystum,

47.08 % (Matanjun, et al., 2009) but higher than that of S. vulgare, 19.43% (Marinho-

Soriano, et al., 2006).

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The variation in the reported composition of Sargassum species may be related to several

environmental factors such as water temperature, salinity, light and nutrients which

influence their ability to stimulate or inhibit the biosynthesis of several compounds (John

et al., 2011; Marinho-Soriano et al., 2006). Most of these environmental factors vary with

the locality and with the season of the year and can be expected to have different values

from one year to another. Furthermore, the concentrations of nutrients are highly

dependent on movements of different layers of water which is influenced by changes in

temperature (Percival & McDowell, 1967).

The holocellulose (cellulose plus hemicellulose) content of the sample was found to be

46.08% (Table 3). The ratio of the alpha-cellulose and the hemicellulose is 0.80. Since the

% hemicellulose content of the sample is higher than alpha-cellulose, this suggests the

need for pretreatment of the sample prior to bioethanol production. Through pretreatment,

hemicellulose components may be solubilized to monomeric sugars such as xylose,

arabinose and galactose thus making cellulose more accessible to enzymatic breakdown.

Based on the proximate and structural analyses, the amount of lignin was estimated to be

less than 1% which is in accordance with what was observed in algal cell wall. It has been

reported that algal cell wall does not contain or nearly contains lignin (John et al., 2011;

Wi et al., 2009). The low lignin content of the sample somehow indicates that less complex

pretreatment and hydrolysis are needed.

3.2 Hydrolysis of Sargassum spp.

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Results showed that the effects of temperature and reaction time on glucose released were

statistically significant (p< 0.05) while the increase in acid concentration from 0.1 % (w/v)

to 1.0 % (w/v) was found to be the least significant at the same confidence level. To

determine the optimum conditions for acid pretreatment and enzyme saccharification of

Sargassum spp. an optimization study was conducted. Statistical analysis also showed

that the t- value of the effect of change in acid concentration from 0.1 to 1.0 % was slightly

above the t-value limit which can be interpreted as possibly significant as opposed to

values above the Bonferroni limit which can be taken with certainty that the effect was

significant. In order to see further the effect of acid concentration on enzyme

saccharification, the acid concentration range used in the optimization study was changed

to 1% as the low level (-) and 5% as the high level (+). On the other hand, the levels for the

two factors, temperature and reaction time, remained the same.

The glucose yield after enzyme sachharification of acid treated biomass is shown in Figure

1(a). The highest glucose released after enzyme hydrolysis was obtained for biomass

treated by both 1% and 5 % H2SO4 at 115 0C for 1.5 h.

The reducing sugar yield was also monitored for each treatment after enzyme

saccharification. The data obtained is shown in Figure 1(b). Four treatments gave high

reducing sugar yield which is around 120 mg/g biomass. The biomass under these

treatments was subjected to high acid concentration, 3-6.4% (w/v) and subjected to more

than an hour of acid pretreatment. It can also be observed that these four treatments were

done at temperatures covering the low level (115 0C), center point (121 0C) and at high

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level (126 0C) in the design. Thus for reducing sugar yield, acid concentration and

hydrolysis time play important roles during pretreatment.

The scanning electron micrograph analysis showed some structural changes in the biomass

before and after being treated with dilute acid and also after enzyme saccharification. The

surface of the untreated biomass was continuous and smooth and coated with crystals.

After treatment it became more rugged and loosed and it seems that more internal

structures were exposed. Based on this result, it is evident that dilute acid pretreatment can

alter the structure of the biomass and may probably increase the accessible surface area for

enzymatic hydrolysis. The images also revealed that degradation happened to the cellulose

fiber after enzyme hydrolysis. The structure of the biomass after enzyme hydrolysis

showed further ruggedness and loosening and more surface area had been exposed. Some

holes were also observed along the surface.

Using the data generated from the enzyme hydrolysis study, the optimum condition for the

acid pretreatment was determined using the response surface methodology. Figure 2

shows the 3D plot generated from Design expert software by fitting the data to a predictive

model. The predictive models generated for glucose and reducing sugar based on actual

factors are as follows:

where
Glucose - glucose concentration, mg glucose/g biomass T- Temperature, 0C

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A- H2SO4 concentration, (w/v) R- Reaction time, h

Based on the optimization results, the optimum acid concentration in terms of glucose

yield was found to be between 2.51 % and 4.01 %( w/v). The optimum temperature and

hydrolysis time were found to be 115 0C and 1.12 -1.5 h, respectively.

Optimization results also revealed that the optimum acid concentration in terms of

reducing sugar yield was found to be between 3.75 % and 4.50 %( w/v) while the optimum

temperature and hydrolysis time were found to be 115 0C and 1.45 -1.5 h. Figures 2c and

2d show the 3D plot generated for reducing sugar yields at hydrolysis time of 1.50 and 0.5

h, respectively. At the lower hydrolysis time level of 0.5 h, the plot is being dominated by

the green region, indicating that an increase in temperature or an increase in acid

concentration did not favor the increase in reducing sugar yield (Figure 2-d). However, at

high hydrolysis time level, 1.5 h, a red region can be seen indicating high reducing sugar

yield.

Based on the predictive model generated, RSM provided optimum conditions if both

reducing sugar and glucose yields were maximized. The optimum conditions for this

criteria were found to be 3.36 to 4.15 % (w/v) H2SO4, 115 0C and 1.44-1.50 h.

It must be worth noting that the optimal pretreatment temperature obtained in this study for

the macroalgae, Sargassum spp. was relatively milder compared to that of terrestrial

biomass. Pretreatment of terrestrial biomass is usually performed under extreme

conditions: high temperature (165- 210 0C), high concentration of chemical solvents or

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long time (4 weeks) due to its high hemicellulose content and presence of lignin (Ge et al.,

2011). Table 4 shows the pretreatment conditions employed for different terrestrial

biomass and that of macroalgae. Although pretreatment at extreme conditions are effective

and high conversion of cellulose from terrestrial plants are possible, it entails high energy

consumption and production cost (Ge et al., 2011). Moreover, at higher severe

pretreatment conditions, it was reported that the glucose released from cellulose may be

converted further to hydromethylfurfural (HMF), which might inhibit the cell growth and

decrease ethanol production (Meinita et al., 2011).

Aside from milder pretreatment temperature, the enzyme saccharification involved in this

study was also done at mild conditions (pH 4.8 and temperature of 50C) which is

reported to have a low utility cost compared to acid or alkaline hydrolysis and does not

have a corrosion problem (Duff and Murray, 1996).

3.3 Effect of enzyme loading and hydrolysis time on enzyme saccharification of

Sargassum spp.

Enzymatic hydrolysis of cellulose is carried out by cellulase enzymes which are highly

specific and the products are usually reducing sugars including glucose (Sun, 2002). In this

study, the pretreated biomass was hydrolysed with cellulases supplemented with -

glucosidase (cellobiase). -glucosidase hydrolyzes the cellobiose which is an inhibitor of

cellulase activity.

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The results of the enzymatic hydrolysis rate study are shown in Figure 3. The glucose

yield was greatly affected by the amount of cellulase added. The increase in cellulase

loading from 5 FPU/g biomass to 50 FPU/g biomass had increased the glucose conversion

to about 15 times at the end of the 96 h of enzyme hydrolysis. High cellulase loading

favored the hydrolysis since more enzymes were available for cellulose breakdown. It was

also observed that for all the systems considered (except for the control), the glucose yield

had reached almost half of the highest peak in just 8 h of hydrolysis. This indicates a faster

hydrolysis rate at the initial stage of the process where more enzymes and cellulose sites

were available for reaction. But as time progresses, the rate of hydrolysis slowed down.

Results showed that the addition of the -glucosidase enzyme indeed prevented the

accumulation of cellobiose for systems, 5 FPU/g biomass & 250 CBU/biomass and 50

FPU/g biomass & 250 CBU/g biomass as shown in Table 5. But in terms of glucose

production, the presence of -glucosidase only improved the hydrolysis of the system with

lower cellulase enzyme loading (5 FPU/g) by about four (4) times. If the cellulase added

was increased by ten times, 50 FPU cellulase/ g biomass, the glucose yield did not

significantly affected by the addition of -glucosidase. At higher cellulase loading, the

cellulase activity seemed to be the dominant factor in the hydrolysis since even in the

presence of cellobiose in the hydrolysate, a known inhibitor of cellulase activity, the

glucose yield was still high.

3.4 Ethanol Fermentation

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The hydrolysate solution obtained from dilute acid pretreatment and enzyme

saccharification using the optimum conditions, 4% H2SO4 (w/v), 115 0C and 1.5 h, was

subjected to fermentation process using Saccharomyces cerevisiae. The initial and final

sugar contents of the samples were determined to monitor the progress of the reaction.

Different factors such as initial pH, temperature and time were considered in the

fermentation process and their effects on ethanol conversion were investigated.

Table 6 shows the amount of ethanol produced using different treatments. It can be noticed

from the data presented that all the glucose present initially in the samples were consumed.

Since each treatment runs started with different initial glucose content, % ethanol

conversion was used to determine the effects of different fermentation factors. Table 6 also

shows the data on % ethanol conversion. Using a two-level factorial design, it was found

out that the three factors; initial pH, temperature and time had significant effects (p < 0.05)

on ethanol conversion. The temperature contributed the highest positive effect indicating

that higher temperature, 40 0C, favored the fermentation. Longer incubation time, 72 h,

gave higher ethanol conversion. However, a negative significant effect was observed for

initial pH, wherein more acidic condition (at pH 4.5) was favorable for ethanol conversion.

Yeast, under anaerobic conditions, metabolizes glucose to ethanol primarily by way of the

Embden-Meyerhof pathway as described in the following net reaction:

C6H12O6 2C2H5OH + 2CO2

The overall net reaction involves the production of 2 moles of ethanol per mole of

glucose, or 510 g of ethanol per kg of glucose, giving a theoretical yield of 51%. From

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Table 6, the % ethanol conversion obtained varies from 65- 89 % depending on the

fermentation conditions used. These values were markedly higher than the theoretical yield

of 51% based on glucose as substrate. Results also indicated that all the glucose present in

the hydrolysate was consumed during fermentation. The discrepancy in the calculated yield

versus the theoretical yield could be attributed to the conversion of other sugars to ethanol.

Material balance revealed that aside from glucose, other reducing sugar was consumed by

the yeast during fermentation.

4.0 Conclusions

Compositional analysis of Sargassum spp. revealed its advantage as feedstock for ethanol

production. The pretreatment temperature obtained for this macroalgae, as well as the

enzyme saccharification conditions employed are relatively milder compared to that of

terrestrial biomass. The ethanol conversions obtained from this study were markedly

higher than the theoretical yield based on glucose as substrate. Since all the glucose present

in the hydrolysate was consumed during fermentation, other sugars were also converted to

ethanol. Therefore, the macroalgae, Sargassum spp., showed significant potential as a

renewable feedstock for the production of bioethanol.

Acknowledgements

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 21

This research was supported by a grant from the Engineering Research and Development

for Technology (ERDT) funded by the Department of Science and Technology of the

Philippine government and conducted at the Department of Agricultural and Biosystems

Engineering, University of Arizona, Tucson, AZ, USA.

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Figure Legends

Figure 1. (a) Glucose and (b) reducing sugar yield after enzyme saccharification of acid
treated Sargassum spp. using 50 FPU cellulase/ g biomass and 250 CBU cellobiase/g
biomass incubated at 50 0C and 100 rpm(Values are expressed as mean standard
deviation, n = 3)
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Figure 2. 3D plots of the glucose and reducing sugar yields for Sargassum spp. after
sulfuric acid pretreatment at different concentrations and reaction time followed by
enzyme saccharification using cellulase(50 FPU/g biomass ) and cellobiase (250 CBU/g
biomass). (a) glucose yield at hydrolysis time = 1.5 h; (b) glucose yield at hydrolysis time
= 0.50 h; ( c) reducing sugar yield at hydrolysis time = 1.5 h; reducing sugar yield at
hydrolysis time = 0.5 h

Figure3. Glucose released during enzyme saccharification of an acid-treated Sargassum

spp. using cellulase and cellobiase incubated at 50 0C and 100 rpm. () 50 FPU
cellulase/g biomass, 0 CBU cellobiase/g biomass ;()50 FPU cellulase/g biomass, 250
CBU cellobiase/g biomass; ()5 FPU cellulase/g biomass, 250 CBU cellobiase/g biomass;
()5 FPU cellulase/g biomass, 0 CBU cellobiase/g biomass; () 0 FPU cellulase/g
biomass, 0 CBU cellobiase/g biomass (Values are expressed as mean standard
deviation, n = 3)

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Table 1. . Experimental design matrix for the optimization of the dilute acid
pretreatment and enzyme saccharification of Sargassum spp.

% Temperature % Temperature
H2SO4 0 H2SO4 0
C C
Treatment Acid Time, Treatment Acid Time,
Run No. (w/w) hr Run No. (w/w) hr
1 T3 5.00 126.00 1.50 25 T10 1.00 115.00 0.50
2 T12 3.00 120.50 0.16 26 T7 3.00 129.75 1.00
3 T11 1.00 115.00 1.50 27 T1 1.00 126.00 0.50
4 T9 5.00 115.00 1.50 28 T10 1.00 115.00 0.50
5 T10 1.00 115.00 0.50 29 T13 3.00 111.25 1.00
6 T7 3.00 129.75 1.00 30 T2 5.00 126.00 0.50
7 T14 0.00 120.50 1.00 31 T12 3.00 120.50 0.16
8 T5 6.36 120.50 1.00 32 T13 3.00 111.25 1.00
9 T7 3.00 129.75 1.00 33 T3 5.00 126.00 1.50
10 T8 5.00 115.00 0.50 34 T4 1.00 126.00 1.50
11 T4 1.00 126.00 1.50 35 T5 6.36 120.50 1.00
12 T6 3.00 120.50 1.84 36 T8 5.00 115.00 0.50
13 T15 3.00 120.50 1.00 37 T15 3.00 120.50 1.00
14 T2 5.00 126.00 0.50 38 T15 3.00 120.50 1.00
15 T3 5.00 126.00 1.50 39 T9 5.00 115.00 1.50
16 T1 1.00 126.00 0.50 40 T6 3.00 120.50 1.84
17 T8 5.00 115.00 0.50 41 T15 3.00 120.50 1.00
18 T2 5.00 126.00 0.50 42 T9 5.00 115.00 1.50
19 T14 0.00 120.50 1.00 43 T5 6.36 120.50 1.00
20 T11 1.00 115.00 1.50 44 T13 3.00 111.25 1.00
21 T12 3.00 120.50 0.16 45 T15 3.00 120.50 1.00
22 T15 3.00 120.50 1.00 46 T1 1.00 126.00 0.50
23 T6 3.00 120.50 1.84 47 T14 0.00 120.50 1.00
24 T11 1.00 115.00 1.50 48 T4 1.00 126.00 1.50

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Table 2
. Proximate analysis of dried Sargassum spp.
% % % % % %
Moisture Ash Crude Crude Crude Total
Fiber Protein Fat Carbohydrates
Seaweed
(Sargassum 11.16 26.19 9.84 10.25 0.75 41.81
spp.) 0.01 0.07 0.07 0.02

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Table 3
Structural composition of Sargassum spp.

% % % %
Alpha- Hemicellulose Holocellulose Mannitol
cellulose

Seaweed 20.35 25.73 46.08 5.04

(Sargassum spp.)

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Table 4

Pretreatment conditions for macroalgae and various terrestrial biomass

Feedstock T , 0C Time H2SO4 References

(min)
Mixed wood 230 0.12 1.17 (w/w, %) Zheng et
(10% birch and 90% al. (2007)
maple)
Wheat straw and aspen 140 60 0.50(v/v) Grohmann
wood et al
(1985)
Olive tree-biomass 170-210 0.2-1.4 w/w, %) Kumar et
al (2009)
Corn stover 180-200 1 0.03-0.06 g acid/g Kumar et
dry biomass al (2009)
Rye straw and bermuda 121 90 1.5 (w/w, %) Sun and
grass Cheng
(2002)
Macroalgae 115 86-90 3.36 - 4.15 (w/v) In this
(Sargassum spp.) study

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Table 5

Amount of cellobiose in the hydrolysate after 96 h of enzymatic saccharification of an acid

treated Sargassum spp.

Enzyme loading*
5 FPU/g 50 FPU/g &
&0 5 FPU/g & 0 CBU/g 50 FPU/g &
CBU/g 250 CBU/g 250 CBU/g
Cellobiose, mg/g
biomass 3.79 0 8.5 0

*cellulase activity in FPU/g biomass & cellobiase activity in CBU/g biomass

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Table 6

Glucose, reducing sugar and ethanol yield at different fermentation conditions using
Sargassum spp. as feedstock
Initial pH Temperature Time, h Glucose, Reducing sugar, Ethanol % Ave
o
C g/Li g/Li Yield*, Ethanol
Initial Final Initial Final g/Li Conversion

4.5 30 48 4.16 0.00 17.21 4.54 2.74 0.03 65.89

6.5 30 48 4.16 0.00 17.21 3.82 2.70 0.04 64.87

4.5 40 48 3.46 0.00 17.65 3.78 2.79 0.09 80.58

6.5 40 48 3.46 0.00 17.65 5.50 2.48 0.09 71.55

4.5 30 72 1.68 0.00 14.32 5.20 1.29 0.02 76.99

6.5 30 72 1.68 0.00 14.32 4.16 1.13 0.02 67.31

4.5 40 72 2.55 0.00 16.47 2.87 2.27 0.04 89.10

6.5 40 72 2.55 0.00 16.47 2.26 2.05 0.02 80.53

*average of triplicate runs

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(a)

(b)

0 0
T1- 1% H2SO4, 0.5 h, 126 C T9- 5% H2SO4, 1.5 h, 115 C
0 0
T2- 5% H2SO4, 0.5 h, 126 C T10- 1% H2SO4, 0.5 h, 115 C
T3- 5% H2SO4, 1.5 h, 126 0C T11- 1% H2SO4, 1.5 h, 115 0C
0 0
T4- 1% H2SO4, 1.5 h, 126 C T12- 3% H2SO4, 0.16 h, 121 C
0 0
T5- 6.4% H2SO4, 1.0 h, 121 C T13- 3% H2SO4, 1.0 h, 111 C
0 0
T6- 3% H2SO4, 1.84 h, 121 C T14- 0% H2SO4, 1.0 h, 121 C
0 0
T7- 3% H2SO4, 1.0 hr, 130 C T15- 3% H2SO4,1.0 hr, 121 C
0
T8- 5% H2SO4, 0.5 hr, 115 C Control- untreated seaweed

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(b)
(a)

(c) (d)

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Highlights

Sargassum spp., have high amounts of carbohydrate and very low lignin content

The pretreatment temperature obtained for the algal biomass is lower compared

with some terrestrial biomass.

The ethanol conversions obtained were markedly higher than the theoretical yield

based on glucose as substrate

Sargassum spp., have the potential as a renewable feedstock for bioethanol

production.

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