Solid acid catalysts pretreatment and enzymatic hydrolysis of macroalgae cellulosic residue for the production of bioethanol

Article abstract of DOI:10.1016/j.carbpol.2015.02.046

The aim of this study is to investigate the technical feasibility of converting macroalgae cellulosic residue (MCR) into bioethanol. An attempt was made to present a novel, environmental friendly and economical pretreatment process that enhances enzymatic conversion of MCR to sugars using Dowex (TM) Dr-G8 as catalyst. The optimum yield of glucose reached 99.8% under the optimal condition for solid acid pretreatment (10%, w/v biomass loading, 4%, w/v catalyst loading, 30 min, 120°C) followed by enzymatic hydrolysis (45 FPU/g of cellulase, 52 CBU/g of β-glucosidase, 50°C, pH 4.8, 30 h). The yield of sugar obtained was found more superior than conventional pretreatment process using H₂SO₄ and NaOH. Biomass loading for the subsequent simultaneous saccharification and fermentation (SSF) of the pretreated MCR was then optimized, giving an optimum bioethanol yield of 81.5%. The catalyst was separated and reused for six times, with only a slight drop in glucose yield.

Full text of DOI:10.1016/j.carbpol.2015.02.046

Accepted Manuscript
Title: Solid acid catalysts pretreatment and enzymatic
hydrolysis of macroalgae cellulosic residue for the production
of bioethanol
Author: Inn Shi Tan Keat Teong Lee
PII:
S0144-8617(15)00162-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2015.02.046
CARP 9719
To appear in:
Received date:
Revised date:
Accepted date:
2-3-2014
24-2-2015
24-2-2015
Please cite this article as: Tan, I. S., and Lee, K. T.,Solid acid catalysts pretreatment and
enzymatic hydrolysis of macroalgae cellulosic residue for the production of bioethanol,
Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.02.046
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Solid acid catalysts pretreatment and enzymatic hydrolysis of macroalgae cellulosic
residue for the production of bioethanol
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Inn Shi Tan, Keat Teong Lee*
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School of Chemical Engineering, Universiti Sains Malaysia, Engineering Campus,
Seri Ampangan, 14300 Nibong Tebal, Pulau Pinang, Malaysia
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* Corresponding author.
Tel.: +604-5996467; Fax: +604-5941013.
E-mail address: ktlee@usm.my
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Highlights
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 Glucose yield of macroalgae cellulosic residue is greatly improved after solid acid
pretreatment.
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 94.6% of the cellulose can be converted into fermentable sugars.
 The catalyst was separated and recycled eight times with slight deactivation.
ABSTRACT
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The aim of this study is to investigate the technical feasibility of converting macroalgae
cellulosic residue (MCR) into bioethanol. An attempt was made to present a novel,
environmental friendly and economical pretreatment process that enhances enzymatic
conversion of MCR to sugars using Dowex (TM) Dr-G8 as catalyst. The optimum yield
of glucose reached 99.8% under the optimal condition for solid acid pretreatment (10%
w/v biomass loading, 4% w/v catalyst loading, 30 min, 120oC) followed by enzymatic
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hydrolysis (45 FPU/g of cellulase, 52 CBU/g of β-glucosidase, 50oC, pH 4.8, 30 h). The
yield of sugar obtained was found more superior than conventional pretreatment process
using H2SO4 and NaOH. Biomass loading for the subsequent simultaneous
saccharification and fermentation (SSF) of the pretreated MCR was then optimized,
giving an optimum bioethanol yield of 81.5%. The catalyst was separated and reused for
six times, with only a slight drop in glucose yield.
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Keywords: Solid acid catalyst; macroalgae cellulosic residue; pretreatment; cellulase;
fermentation; bioethanol
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1. Introduction
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The world is currently facing the dilemma of high crude-oil price while
simultaneously energy consumption keeps on increasing due to increasing world’s
population and rapid economic growth led by industrialization (Katinonkul et al., 2012).
In addition, the emission of greenhouse gas (GHG) due to fossil fuels consumption has
caused climate change. Considering these issues, it is now inevitable to shift toward
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renewable sources of energy. Bioethanol production from biomass has gained
considerable interest as a source of renewable transportation fuel. The production of first-
generation bioethanol from food-based biomass is not sustainable because it competes
with food sources (Nigam & Singh, 2011). Therefore, the focus on bioethanol has shifted
to the development of second-generation bioethanol, by utilizing lignocellulosic biomass.
Although second-generation bioethanol is attractive because it is made from non-edible
feedstock, but the difficulty in separating lignin from lignocelluloses has impeded the
commercialization potential of this renewable source (Karthika, Arun & Rekha, 2012).
Now, macroalgae has emerge as the third-generation biomass that can be used in
bioethanol production. Among the advantages of using macroalgae as feedstock is it does
not need land and freshwater for their cultivation, it grows quickly, and is lignin free
(Goh & Lee, 2010).
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The red macroalgae, Eucheuma cottonii, is widely cultivated in Malaysia, and is
used as biomass for the production of κ-carrageenan. The major polysaccharide
constituents of red macroalgae are κ-carrageenan, which are the most commercially
70 important polysaccharides for red macroalgae. κ-carrageenan can be easily obtain from
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red macroalgae through extraction or dissolving them into an aqueous solution (Chan et
al., 2013). After extracting κ-carrageenan, a huge amount of residual materials are left
behind, which is usually called macroalgae cellulosic residue (MCR). The residual
biomass can be utilize for the production of bioethanol because it contains large amount
of cellulose and with low lignin content. The utilization of MCR as an energy resource
would pave the way for converting waste material to a product that has high commercial
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value. In general, bioethanol production from biomass involves pretreatment, enzymatic
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pretreated prior to enzymatic hydrolysis for bioethanol production.
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The advantage of pretreating cellulosic materials prior to hydrolysis have been
well established in the literature (Ahmed et al., 2013b; Kootstra et al., 2009). However,
the pretreatment process of macroalgae for the production of bioethanol is still at its
infancy stage. The establishment of an efficient pretreatment method in order to facilitate
the conversion of sugars during the enzymatic hydrolysis is the key step for bioethanol
production (Monavari, Galbe & Zacchi, 2009; Schultz-Jensen et al., 2013). The goal of
pretreatment process is to reduce the crystallinity of cellulose, increase the porosity of the
cellulosic materials and thus enhance the enzymatic hydrolysis productivity (Cabiac et al.,
2011). Different examples of bioethanol production and pretreatment methods for
macroalgae have been described in the literature. One study reported that carbohydrates
from Laminaria japonica can be effectively hydrolysed to simple sugars by dilute H2SO4
treatment (Ge, Wang & Mou, 2011). Another study reported that dilute acid pretreatment
of Saccharina japonica can improve the efficiency of enzymatic hydrolysis followed by
simultaneous saccharification and fermentation (SSF) (Lee et al., 2013). Although
sulfuric acid are powerful agent for cellulose pre-treatment, however, it has its
disadvantages. Sulfuric acid are toxic, corrosive, hazardous, and thus require reactors that
are resistant to corrosion, which makes the pretreatment process very expensive. In
addition, it is also very difficult to recycle the catalyst and its disposal would require
proper waste water treatment facilities. As an alternative to dilute acid pretreatment
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method, there is the possibility of using heterogeneous solid acid catalyst which can
overcome the above drawbacks (Chareonlimkun et al., 2010). Recently, several solid
acids are reported in the literature as efficient catalytic systems for the hydrolysis of
cellulose, starch and other polysaccharides (Marzo, Gervasini & Carniti, 2012; Ormsby,
Kastner & Miller, 2012; Shen et al., 2013; Yamaguchi & Hara, 2010). However, to the
best of our knowledge, very little information is available on the use of solid acid catalyst
for the pretreatment of cellulosic biomass from macroalgae for the production of
bioethanol.
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Thus, the objectives of this study are to, (1) optimize the pretreatment conditions
for macroalgae cellulosic residue (MCR) using solid acid catalyst, Dowex (TM) Dr-G8
base on the highest glucose yield that were obtained through enzymatic hydrolysis of the
pretreated MCR; (2) study the effect of enzyme loading on the enzymatic hydrolysis of
pretreated MCR; (3) study the effect of pretreated MCR loading on bioethanol production
through simultaneous saccharification and fermentation (SSF) by Saccharomyces
cerevisiae.
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2. Methods
2.1. Raw materials and chemicals
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Eucheuma cottonii were obtained from Futt Put Enterprise (north coast of Sabah,
Borneo). The macroalgae was first washed with tap water to remove impurities and dried
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until the algae disintegrated. Subsequently, the κ-carrageenan extract was filtered (45 µm
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mesh size). The extract residue was boiled for 30 min with 2 L of distilled water and
filtered with pressure pump. The macroalgae cellulosic residue (MCR) was then dried at
50oC, pulverized, screened through 80 mesh and used for subsequent experiments.
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The enzymes used in the enzymatic hydrolysis were commercial cellulase
(Celluclast 1.5L, Novozyme, Denmark) and β-glucosidase (Novozyme 188, Novozyme,
Denmark), which were all purchased from Science Technics Sdn. Bhd. The activities of
cellulase and β-glucosidase were reported by the manufacturer as 82.08 filter paper unit
(FPU)/mL and 326.12 cellobiase unit (CBU)/mL, respectively. Strong acid cation-
exchange resin Dowex (TM) Dr-G8, calcium hydroxide, standard D-glucose, hydroxyl-
129 methyl furfurals, Saccharomyces cerevisiae (YSC2, type II), yeast extract, peptone,
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dextrose, and bicinchoninic acid (BCA) kit were purchased from Sigma-Aldrich (USA).
Table 1 shows the properties of Dowex (TM) Dr-G8 catalyst. Acetic acid, sodium acetate,
sodium hydroxide, sulfuric acid, hydrochloric acid and ethanol were purchased from
Fisher Scientific (UK).
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All the chemicals used in this study were analytical grade and all the experiments
were performed in triplicates and the results are presented as mean ± standard deviation.
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2.2. Yeast cultivation
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For the inoculum preparation of Saccharomyces cerevisiae (YSC2, type II), dry
yeast was dispersed in sterile YEPD medium (1% (w/v) yeast extract, 2% peptone, 2%
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dextrose) at a concentration of 1.75% (w/v) in a 250 mL Erlenmeyer flask at pH 5.0. The
141 preculture was incubated at 35oC for 24 h in a shaking incubator (Benchmark Scientific
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Inc., New Jersey) at 120 rpm. Yeast cells were harvested by centrifugation (10, 000 × g,
15 min), suspended in sterilized water and used as inoculum in the SSF process.
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2.3. Chemical compositional analysis of macroalgae cellulosic residue (MCR)
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The cellulose, lignin and hemicellulosic fractions of pulverized macroalgae
cellulosic residue (MCR) were determined according to a modified method that is based
on the National Renewable Energy Laboratory (NREL, Golden, CO) analytical methods
(Sluiter et al., 2008). Approximate 300 mg of MCR was initially subjected through a
primary 72% sulfuric acid hydrolysis at 30oC for 60 min. In the second step, the reaction
151 mixture was diluted to 4% sulfuric acid and autoclaved at 121oC for 1 h. The content of
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sugar was quantified with high performance liquid chromatography (HP-LC). The
remaining acid-insoluble residue is considered as acid-insoluble lignin (AIL).
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2.4. Pretreatment of MCR
2.4.1. Solid acid catalyst pretreatment
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The macroalgae cellulosic residue (MCR) was pretreated using Dowex (TM) Dr-
G8. The effects of the following parameters on the pretreatment of MCR were
investigated: amount of MCR (8-14%, w/v), amount of Dowex (TM) Dr-G8 (0-6%, w/v),
pretreatment time (15-60 min) and temperature (110-140oC). The liquid amount was
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fixed at 50 mL, and pre-determined amount of dried MCR were added at different S/L
ratios. The mixture was then mixed with different amount of Dowex (TM) Dr-G8 and
incubated at different temperature. The pretreatment was conducted in an autoclave
reactor with gentle mixing at a speed of 320 rpm while the internal pressure of the
autoclave reactor was kept constant at 10 bars. After a specific pretreatment time, the
solid and liquid fractions were separated by filtration using filter paper. Subsequently, the
residual substrates were washed and neutralized with distilled water and then dried at
50oC until attain constant weight. The pretreated MCR was then used as the substrate for
enzymatic hydrolysis.
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2.4.2. Dilute acid and alkali pretreatment
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The effectiveness of using Dowex (TM) Dr-G8 as catalyst for pretreatment was
evaluated by comparing with conventional dilute acid and alkali pretreatment method.
For dilute acid pretreatment method, 10% (w/v) MCR was treated with 1% (w/v)
sulphuric acid at 120oC for 30 min. After that, the residue solid material was separated
and washed with excess distilled water until the washed water had a pH of 5.5. The
sample was then dried in an oven at 50oC until achieving constant weight. The solid
fraction was used as the substrate in the subsequent enzymatic hydrolysis step.
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For alkali pretreatment method, 10% (w/v) MCR was treated with 1% (w/v)
178 NaOH at 120oC for 30 min. After that, the pretreated substrate was filtered by using filter
179 paper and washed with dilute acid solution (HCl) until pH 5.5 was attained. The sample
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was then dried in an oven at 50oC until achieving constant weight. The solid fraction was
used as the substrate in the subsequent enzymatic hydrolysis step.
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2.5. Enzymatic hydrolysis
2.5.1. Enzymatic hydrolysis of pretreated macroalgae cellulosic residue (MCR)
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To evaluate the effectiveness of MCR pretreatment at various conditions, the
pretreated MCR was subjected to enzymatic hydrolysis with 2% (w/v) solid loading and
15 FPU/g of cellulase and 52 CBU/g of β-glucosidase in accordance with National
Renewable Energy Laboratory (NREL)’s Chemical Analysis and Testing Standard
Procedure no. 009 (NREL, 2004). For the enzymatic hydrolysis of pretreated MCR, a
reaction mixture containing pretreated MCR, cellulase, β-glucosidase, and 50 mM
sodium acetate buffer (pH 4.8) was incubated in a shaking incubator at 50oC and 120 rpm
(Tan & Lee, 2014). Samples were taken periodically and in order to terminate the
enzymatic activity, samples were boiled for 15 min immediately after each sampling. The
residues were then separated from liquid by centrifugation (10, 000 × g, 5 min). The
supernatants were filtered and preserved at -2oC and were analyzed for reducing sugar
analysis to determine the percentage of hydrolysis by high-performance liquid
chromatography (HP-LC).
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2.5.2. Effect of cellulase concentration on enzymatic hydrolysis of pretreated MCR
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Using the optimum pretreatment conditions obtained from the previous section
(biomass loading= 10% (w/v), catalyst loading= 4% (w/v), pretreatment time= 30 min,
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and temperature= 120oC), pretreated MCR is subsequently used to investigate the effects
of cellulase concentration on enzymatic hydrolysis of pretreated MCR. The cellulase
concentrations studied are 5, 15, 30, 45 and 60 FPU/g while the β-glucosidase
concentration were maintained constant at 52 CBU/g for all experiments. The solid
loading and reaction time were fixed at 2% (w/v) and 72 h while other conditions were
similar as reported in Section 2.5.1.
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2.5.3. Determination of cellulase adsorption on cellulose
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Cellulase adsorption during the enzymatic hydrolysis process was measured in a
250 mL Erlenmeyer flask with 2% (w/v) for MCR pretreated with different methods
209 (Dowex (TM) Dr-G8, H2SO4 and NaOH). The dosage of cellulase for all the hydrolysis
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experiments is 45 FPU/g while other conditions were similar as reported in Section 2.5.1.
Samples were withdrawn after 2 h of enzymatic hydrolysis and centrifuged at 10,000 × g
for 5 min to remove insoluble materials. The supernatant obtained was analyzed for
reducing sugar and protein content. The protein content of the supernatant (free cellulase)
was determined using bicinchoninic acid (BCA) kit from Sigma Aldrich (Section 2.7.2).
The amount of cellulase bound to the cellulose was calculated by subtracting the amount
of free cellulase in the supernatant from the amount of cellulase initially added to the
reaction medium (Pierre et al., 2011).
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2.5.4. Recycling of catalyst
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The possibility of re-using the Dowex (TM) Dr-G8 catalyst was determined by
running pretreatment process at 120oC for 30 min with solid loading of 4% (w/v) for
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repeated cycles. After completing the first cycle of pretreatment, the solid acid catalyst
mixed with the pretreated MCR was filtered. The resulting brown color solid residue was
vigorously stirred in 250 mL of distilled water for 20 min and left standing for a while.
Subsequently the catalyst will settle down to the bottom of the beaker while the tiny
pretreated MCR will remain suspended in the solution. The solid acid catalyst was then
easily recovered by decantation. The collected solid acid catalyst were dried at room
temperature for 24 h and reused for another cycle of pretreatment. This was repeated for
6 cycles. For the enzymatic hydrolysis condition, pretreated MCR (2% w/v) was
hydrolysed for 24 h in sodium acetate buffer (50 mM, pH 4.8) at 50oC using a 250 mL
Erlenmeyer flask. The enzyme loading used were 45 FPU/g of cellulase and 52 CBU/g of
β-glucosidase.
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2.6. Simultaneous saccharification and fermentation (SSF)
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After establishing the optimal enzyme loading (Section 2.5.2), 4-10 % (w/v)
concentration of pretreated MCR was used for simultaneous saccharification and
fermentation (SSF) process to determine the suitable biomass concentration. S. cerevisiae
was used for fermentation. Pretreated MCR was prehydrolyzed for 24 h in sodium acetate
buffer (50 mM, pH 4.8) at 50oC using a 250 mL Erlenmeyer flask. The enzyme loading
used were 45 FPU/g of cellulase and 52 CBU/g of β-glucosidase. The temperature was
subsequently reduced to 43oC for further 8 h to allow for SSF after yeast inoculation. SSF
experiments were performed in a shaking incubator operated at 120 rpm and was adjusted
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to pH 5.0 by 5 M NaOH. The flasks were sealed with rubber stoppers equipped with
needles for CO2 venting. 1.5 mL of sample was withdrawn at different interval time
during SSF and was centrifuged at 10, 000 × g for 15 min. The supernatant was stored in
-2oC freezer and subjected for sugar and bioethanol analysis.
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2.7. Analytical methods
2.7.1. Sugar, by-product and bioethanol analysis
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Supernatants were analyzed for soluble sugar and by-product content using
Agilent series 1200 infinity high-performance liquid chromatography (HPLC). The
HPLC system was equipped with a 385-ELSD (evaporative light scattering detector) and
a Hi-Plex Ca column (300 × 7.7 mm). Distilled-deionised water was used as the mobile
phase with a flow rate of 0.6 mL/min and injection volume of 20 µL. Purified nitrogen
was used as carrier gas (70 psi) for the detector. The HPLC-ELSD’s spray chamber
temperature was set at 40oC whereas detector temperature at 80oC. Prior to injection,
256 samples were diluted 50 times with deionized water and filtered with 0.20 μm syringe
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filter (Nylon membrane, Fisher Scientific). The identities of the components were
authenticated by comparing their retention times with those of pure compounds (Sigma-
Aldrich, USA). The glucose content was calculated according to calibration curves
plotted with standard glucose. The sugar yield was calculated as:
Concentration (g/L) of glucose at time of t
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Yield % 
100%
(1)
Initial concentration (g/mL) of substrate
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The bioethanol produced during the SSF process was quantified by gas
chromatography (GC) using a 5890 Series II chromatography equipped with flame
ionization detector (FID) (Hewlett Packard, Palo Alto, CA) . The column used was 2.0 m
in length and 0.2 cm I.D, 80/120 mesh Carbopack B-DA/4% Carbowax 20M (Supelco,
USA). The temperature of the injection unit and detector were 225oC. The oven was
heated to 100oC for 2 min and the temperature was raised to 175oC at a rate of 10oC/min.
Helium was used as the carried gas while 0.5% (v/v) 2-Pentanone was used as internal
standard. Samples of 2 µL were injected. The bioethanol yield, expressed as percentage
of the maximum theoretical yield that can produced from pretreated MCR, was calculated
using the following equations (Keating et al., 2004):
(2)
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where [EtOH]f is the bioethanol concentration at the end of the fermentation (g/L); and
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term “0.51 × f × [Biomass] × 1.111” corresponds to the theoretical bioethanol
concentration, where [Biomass] is the dry biomass weight concentration at the beginning
277 of the fermentation (g/L); f is the cellulose fraction of dry biomass (g/g); 0.51 is the
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conversion factor for glucose to bioethanol based on the stoichiometric biochemistry of
yeast and 1.111 is the conversion factor for cellulose to equivalent glucose.
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2.7.2. Determination of protein concentration
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The solution obtained from Section 2.5.3 was diluted to 2 mL using 50 mM
sodium acetate buffer. After centrifuging the solution at 10, 000 × g for 2 min, 1 mL of
the diluted supernatant was transferred to a test tube, and 1 mL of the QuantiPro Working
Reagent (bicinchoninic acid reagent) was added. The test tube was vortex gently for
thorough mixing, left standing for 1 h in water at 60oC and then cooled to room
temperature. The absorbance of the samples was measured at 562 nm using a
spectrophotometer (Agilent Technologies carry 60 UV-Vis). The protein concentration of
the samples was calculated from a bovine serum albumin (BSA) standard curve.
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2.7.3. Scanning electron microscopy (SEM) analysis
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The morphology and the physical structure of the untreated MCR and the MCR
treated with solid acid catalyst were observed by scanning electron microscopy (SEM)
292 using a Fei Quanta 450 FEG (Eindhoven, NL). The samples were dried at 50oC to
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constant weight and coated with Au/Pd film. All images were taken at a magnification of
10000× and observed using a voltage of 5 kV.
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2.7.4. Brunauer-Emmett-Teller (BET)
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The Brunauer-Emmett-Teller (BET) surface area, pore size and pore volume of
the untreated and treated MCR was determined using nitrogen adsorption/desorption
299 isotherms at -196oC in a surface area analyzer (ASAP 2020, Micromeritics Co., USA).
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Prior to analysis, the sample was degassed for 8 h at 120oC under vacuum (5 mmHg) to
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remove moisture and any contaminants. BET surface area was calculated from these
isotherms using the BET method. Total volume of pores was determined by single point
adsorption total pore volume of pores with P/Po at 0.984. The average pore size was
calculated by Density Functional Theory (DFT) method (Seaton, Walton & quirke, 1989)
and software provided by Micromeritics Instrument Corporation.
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2.7.5. Ammonia temperature programmed desorption (NH3-TPD)
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The acidity of the solid acid catalysts were measured by temperature
programmed desorption of ammonia (NH3-TPD) using Autochem II 2920 chemisorption
analyser, (Micromeritics Instruments, USA) equipped with thermal conductivity detector
310 (TCD). 0.054 g catalyst was placed in an adsorption vessel and heated to 450oC in He
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flow for 1 h with a rate of 5oC/min. Subsequently it was cooled to 100oC in He flow and
15% NH3 in He was passed through the sample for 1 h. NH3 desorption was conducted
from 100 to 600oC with heating rate of 5oC/min under He flow.
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3. Results and discussion
3.1. Macroalgae cellulosic residue (MCR) composition
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Cellulose and acid insoluble lignin were analysed using the method of two-step
acid hydrolysis. The results showed that cellulose fraction comprised 68% and there was
no acid insoluble lignin detected in the macroalgae cellulosic residue biomass. The HP-
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LC analysis of 72% sulfuric acid hydrolysate showed that glucose was the only main
component of MCR. The high carbohydrate content of MCR makes it a very promising
substrate for bioethanol production.
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3.2. Pretreatment of macroalgae cellulosic residue (MCR)
3.2.1. Effect of biomass loading
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The effect of macroalgae cellulosic residue (MCR) loading on the solid-acid
pretreatment process was studied. Fig. 1 shows the enzymatic hydrolysis yield at 120oC,
4% (w/v) Dowex (TM) Dr-G8 and 30 min pretreatment condition. Up to 24 h of
enzymatic hydrolysis, there were no significant differences in glucose yield at all levels
of biomass loading. However, after 24 h, it can be clearly seen that the glucose yield for
biomass loadings with 8% (w/v) and 10% (w/v) are significantly higher than biomass
loadings of 12% (w/v) and 14% (w/v). In fact for biomass loadings of 12% (w/v) and
14% (w/v), a slight drop in glucose yield was observed after 48 h. This indicated that the
increase of biomass loading in the pretreatment process does not enhance the enzymatic
hydrolysis of the pretreated MCR to some extent. When the biomass loading was
increased higher than 10% (w/v), it became hard to keep the reaction system in
homogeneous form because of insufficient liquid, which resulted to a slurry with high
viscosity that is difficult to handle (Kim, Lee & Jeong, 2014). Besides that, when the
biomass loading was too high, the MCR could not interact sufficiently with the solid acid
catalyst, resulting to low glucose yield. Similar results were also reported by other
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researchers in which the optimum Gelidium amansii content for H2SO4 acid pretreatment
were 10% (Ra et al., 2013). Therefore, the biomass loading of 10% (w/v) was applied in
further experiments.
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3.2.2. Effect of catalyst loading
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In this section, solid acid pretreatment experiments of macroalgae cellulosic
residue (MCR) was carried out at 120oC, pretreatment time of 30 min and by varying the
catalyst loading from 0% to 6% (w/v). The pretreated MCR were then subjected to
enzymatic hydrolysis and the content of glucose released was detected to evaluate the
effectiveness of the pretreatment using Dowex (TM) Dr-G8. With regard to the
environmental impact and operating cost, the lowest catalyst loading that can give the
best hydrolysis performance would be desirable (Ho et al., 2013). Fig. 2 illustrates the
effect of catalyst loading on pretreatment of MCR. It can be seen that the yield of glucose
generally increases with time for all catalyst loading because longer hydrolysis time will
allow more enzyme to depolymerize cellulose to glucose. Apart from that, the results also
showed that the yield of glucose formation was significantly affected by the amount of
catalyst loading. For example, when no catalyst was used in the pretreatment step, the
highest glucose yield achieved at the 24 h of hydrolysis time was only 66%. However,
when the catalyst loading was increased to 2% (w/v) and subsequent 4% (w/v), the
glucose yield increased to 71.8% and 74.9%, respectively. Higher loading of Dowex (TM)
Dr-G8 accelerates the pretreatment hydrolysis rate of MCR, which can be explained due
to the increase in the total number of active catalytic sites available for the reaction
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(Rinaldi, Palkovits & Schüth, 2008; Tan, Lam & Lee, 2013). Besides that, pretreatment
could also help to reduce the crystallinity of cellulose by removing hydrogen linkages by
solid acid hydrolysis that led to rapid and efficient enzymatic hydrolysis of MCR. In
addition, no furfural or hydroxyl-methyl furfurals (HMF) were detected in the solid acid
pretreated MCR hydrolysates for catalyst loading below 4% (w/v). On the other hand,
there was a decreasing trend for glucose production when the loading of solid acid
catalyst was increased above 4% (w/v) in which the yield dropped to 59.6%. This means
that increasing the catalyst dosage beyond the optimal value resulted in a decrease of
glucose yield. The main reason for this reduction would most probably be due to
excessive acidity, which cause the degradation of the released sugars into unwanted side
products such as furfurals and hydroxyl-methyl furfurals (Gupta, Khasa & Kuhad, 2011;
Park et al., 2011). Thus a solid acid catalyst loading of 4% (w/v), which provided the
highest glucose yield, was selected as the optimal catalyst loading for subsequent
experiments in this study.
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3.2.3. Effect of pretreatment time
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The pretreatment time is another important factor that have significant effect on
the productivity of glucose. The effect of pretreatment time (15-60 min) for MCR (10%
w/v) pretreated with Dowex (TM) Dr-G8 (4% w/v) at 120oC and subsequent enzymatic
hydrolysis (50oC, pH 4.8, 72 h) is presented in Fig. 3. At 48 h of hydrolysis time, the
yield of glucose was found to increase from 97.6% to 99.8% when the pretreatment time
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was increased from 15 min to 30 min. This is because longer pretreatment duration
allows sufficient time for the solid acid catalyst to degrade MCR biomass and thus more
cellulose site is available for enzymatic hydrolysis. However, further increase in
pretreatment time gave a negative effect on the glucose yield. The glucose yield decrease
to 82.8% (48 h hydrolysis time) when the pretreatment time was prolonged to 60 min.
This is because longer pretreatment time might degrade the cellulose and release soluble
glucose from MCR during the pretreatment process itself, which led to less amount of
cellulose that is available for enzymatic hydrolysis and hence a decrease in glucose yield.
This finding is supported by Harun and Danquah (2011), where they reported that
increasing pretreatment time up to 60 min does not increase the hydrolysis process of
microalgae but instead pose a risk in reducing the production of bioethanol. Besides that,
shorter duration for pretreatment process is actually favourable because it has a positive
impact on energy consumption. Therefore, 30 min of pretreatment time was chosen as the
optimum value for treating macroalgae cellulosic residue (MCR). (Harun & Danquah,
2011)
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3.2.4. Effect of pretreatment temperature
The effect of pretreatment temperature on the enzymatic hydrolysis is presented
401 in Fig. 4. When the pretreatment temperature was increased from 110 to 120oC, the
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glucose yield of pretreated macroalgae cellulosic residue (MCR) improved significantly.
This shows that MCR pretreated at higher temperature was more acquiescent to
enzymatic hydrolysis. After 48 h of enzymatic hydrolysis, the glucose yield attained was
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405 99.8% after being treated at 120oC, which increased over 7.2% in comparison with that at
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110oC. This phenomenon could be explained as high pretreatment temperatures facilitate
cellulose fibers to dissolve faster and making more cellulose accessible to enzyme attack.
In other words the adsorption rate of enzyme onto the macroalgae cellulosic residue
surface increased at higher pretreatment temperature which leads to higher hydrolysis
rate. However, when the pretreatment temperature was increased more than 120oC, the
glucose yield decreased significantly. For example, at 140oC pretreatment temperature,
the glucose yield obtain was only 66.5% even at the longest hydrolysis time. This is again
possibly due to partial cellulose degradation during pretreatments under harsh conditions.
414 Harun and Danquah (2011) also reported that acid pretreatment temperature above 140oC
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gave a significant decrease in the amount of reducing sugars from microalgae biomass.
Therefore, 120oC is the optimum pretreatment temperature and it is worthwhile to note
that this optimum temperature is relatively milder compared to that of terrestrial biomass
(Jung et al., 2013; Tucker et al., 2003). Up to this point, the optimum pretreatment
condition for macroalgae cellulosic residue (MCR) is 4% (w/v) Dowex (TM)-Dr G8,
120oC, and 30 min. At this condition, no degradation of cellulose was detected.
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3.3. Enzymatic hydrolysis
3.3.1. Effect of enzyme loading
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For evaluation on the effect of enzyme loading on MCR pretreated with Dowex
(TM) Dr-G8, samples were subjected to enzymatic hydrolysis with 5-60 FPU/g of
425 cellulase supplemented with β-glucosidase. The reason for adding β-glucosidase is to
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hydrolyze cellobiose which is an inhibitor to cellulase activity. MCR pretreated at 120°C
for 30 min with 4% (w/v) Dowex (TM) Dr-G8 were used as starting materials for this
part of study and the results are shown in Fig. 5. When the hydrolysis time is below 36 h,
increasing the cellulase loading from 5 to 30 FPU/g seems to have a significant increase
in the glucose yield. However further increasing the cellulase loading beyond 30 FPU/g
has little effect on the glucose yield. This finding can be easily justified as follow. High
cellulase loading will increase the availability of enzymes and therefore more cellulase is
adsorbed onto the substrate surface for reaction to occurs (Ran et al., 2012). However,
increasing the cellulase loading beyond a certain value does not have significant effect on
the glucose yield because the limiting factor has shifted from cellulase loading to β-
glucosidase which was fixed at 52 CBU/g for all experiments. To explain further, initially
437 cellulase will hydrolyze cellulose to cellobiose and subsequently β-glucosidase will
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hydrolyze cellobiose to glucose. Thus, initially, when cellulase loading was increased,
more cellobiose will be obtained and being converted to glucose by β-glucosidase. This
will go on until there is too much cellobiose that now the limiting factor is the amount of
β-glucosidase that was fixed at 52 CBU/g. In other words, the amount of β-glucosidase is
not sufficient for effective hydrolysis of cellobiose into glucose at higher cellulase
loading causing an accumulation of cellobiose. Cellobiose is a dimer of glucose that has a
strong inhibition toward endo- and exoglucanases (from cellulase) that can significantly
slows down the entire enzymatic hydrolysis process (Ruangmee & Sangwichien, 2013;
Teugjas & Väljamäe, 2013). Besides that, high cellulase loading could also reduce the
absorption efficiency of the enzyme on cellulose due to high viscosity that could also
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contribute to a lower glucose yield (Singh et al., 2009; Zheng et al., 2009). This was
supported by the findings shown in Fig. 5 whereby at hydrolysis time of 30 h, glucose
yield decreased by 14.4% when the cellulase loading was increased from 45 to 60 FPU/g.
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In short, either too low or too high enzyme loading is not appropriate for
enzymatic hydrolysis. However, minimizing enzyme consumption is an important way to
reduce cost. Thus, the optimal loading of cellulase was determine as 45 FPU/g which will
be used for subsequent experiments. The optimum enzyme dosage was found lower as
compared to acid or alkaline hydrolysis and does not pose any corrosion problem
(Borines, de Leon & Cuello, 2013).
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3.3.2. Effect of different pretreatment methods
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In order to further understand the digestibility of the macroalgae cellulosic residue
(MCR) pretreated with Dowex (TM) Dr-G8, MCR was also pretreated with two other
methods (H2SO4 and NaOH) and the content of protein in the supernatant and glucose
yield were determined, as summarized in Table 2. It is well established that the
adsorption of cellulase on substrate is a prerequisite for enzymatic hydrolysis of cellulose
to occur. Among the different pretreated substrates evaluated in this study that were
subjected for enzymatic hydrolysis, the MCR pretreated with Dowex (TM) Dr-G8 were
observed to be more vulnerable to enzymatic hydrolysis and gave the highest glucose
yield (99.8%). Therefore, it could be speculated that for the same cellulase loading, MCR
pretreated with Dowex (TM) Dr-G8 would adsorb significantly more cellulase than MCR
pretreated with H2SO4 or NaOH. This is supported by the fact that only 19.6% protein
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(cellulase) remained in the supernatant of the MCR pretreated with Dowex (TM) Dr-G8,
which was significantly lower than that in the supernatant of the MCR pretreated with
471 H2SO4 (22.3%) or NaOH (25.3%), respectively. This result proved the speculation that
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MCR pretreated with Dowex (TM) Dr-G8 was able to adsorb more cellulase at the
beginning of the enzymatic hydrolysis than the MCR pretreated with H2SO4 or NaOH.
This is probably because Dowex (TM) Dr-G8 act as a swelling agent, which enhances the
surface area of the substrate and make the substrate more amenable for enzymatic action
and better digestibility. This comparison shows that Dowex (TM) Dr-G8 is a far more
superior catalyst than NaOH and slightly better than H2SO4. The role of Dowex (TM) Dr-
G8 resins is merely an acidifier of the aqueous slurries, having a similar effect as an
479 aqueous solution of H2SO4 and therefore it is expected that the mechanism of cellulose
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depolymerisation using Dowex (TM) Dr-G8 is similar to the depolymerisation
mechanism with H2SO4 (Watanabe, 2010). In contrast, the MCR pretreated with NaOH
when subjected to enzymatic hydrolysis resulted to a sugar yield of only 80.2%. As
compared to MCR pretreated with Dowex (TM) Dr-G8, the lower enzymatic hydrolysis
efficiency in MCR pretreated with NaOH might be due to lower digestibility.
Furthermore, the advantage of using alkali pretreatment is that it can efficiently remove
lignin from the lignocellulosic biomass, which was not applicable in this case as MCR
does not contain lignin (Dagnino et al., 2013; Sambusiti et al., 2013). Besides that,
Dowex (TM) Dr-G8 is more environmental friendly because it can be easily separated
from the biomass after the pretreatment process and can be reuse. Additional experiments
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were conducted to test the possibility of recycling Dowex (TM) Dr-G8 cation-exchange
resin and will be presented in the section 3.3.3.
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A mass balance of the process (from pretreatment to enzymatic hydrolysis) was
summarized as shown in Fig. 6. The remaining solid fraction was separated from the
hydrolysate prior to enzymatic hydrolysis. On the basis of 100 g of MCR, about 80-81 g
of pretreated MCR can be recovered after pretreatment. Subsequently, about 0.77, 1.08
and 0.05 g glucose can be recovered in the prehydrolysate from solid acid, dilute acid,
and sodium hydroxide pretreatment, respectively. The pretreated MCR was subjected to
enzymatic hydrolysis with a buffer solution that comprising of 45 FRP/g of cellulose, 52
CBU/g of β-glucosidase and incubated at 50oC for up to 30 h. Finally, the mass balance
demonstrates that about 80.6, 80.0, and 64.9 g glucose can be recovered from enzymatic
hydrolysis of solid acid, dilute acid, and sodium hydroxide pretreated MCR for the
conditions selected, respectively.
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3.3.3. Reuse of catalyst
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In order to make the entire process more attractive and industrially viable, the
catalyst must have a long life-usage and consistently give high activity. Catalyst
recyclability study were performed with 4% (w/v) of Dowex (TM) Dr-G8 at 120oC and
30 min pretreatment time, since these conditions resulted in the highest glucose yield
during enzymatic hydrolysis. As shown in Fig. 7, the glucose yield maintained at around
94% till the fifth run, with only slight decrease during the sixth run. The results showed
that the catalyst was not significantly deactivated even after repeated use. This means that
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the functional groups of the solid acid catalyst remained intact even after repeated use
and the resin can be used repeatedly by simply washing with distilled water. Therefore, it
can be concluded that the catalyst is very stable even after being used repeatedly for six
times.
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3.4. Simultaneous saccharification and fermentation (SSF)
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In the previous section, the effect of pretreatment using Dowex (TM) Dr-G8 was
only evaluated up to the stage of glucose yield. Thus, in this section, the use of SSF
process for the production of bioethanol from MCR pretreated with Dowex (TM) Dr-G8
will be presented. For any bioethanol from biomass production process, it is important to
produce high concentration of bioethanol due to the energy intensive nature of distillation
and dehydration process (Ahmed et al., 2013a). In order to achieve a high final
bioethanol concentration, a high substrate loading, and hence a high cellulose content, is
crucial for the SSF process to be economically viable. The effect of solid loading
(pretreated MCR) on bioethanol production using the SSF process with prehydrolysis
was carried out at 43oC and the results are shown in Fig. 8. It is important to differentiate
between conventional SSF and SSF process with prehydrolysis. SSF with prehydrolysis
has the potential to significantly improve the bioethanol yield because this will facilitate
more effective stirring at the beginning of the process (Hoyer, Galbe & Zacchi, 2013).
Fig. 8 indicates that the glucose accumulated during prehydrolysis was rapidly consumed
after the inoculation of S. cerevisiae. In general, the glucose concentration accumulated
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during prehydrolysis increased with the higher solid loading, 37.8, 65.3, 85.5, and 110.6
g/L corresponding to solid loading of 4%, 6%, 8% and 10% (w/v), respectively. The
glucose concentration was found to decrease as the SSF process progressed which was
accompanied by a rapid increase in the fermentation product. The glucose was totally
consumed within 3 h after inoculation and then remained at a low level for all solid
loading, indicating that fermentation by the yeast was much faster that enzymatic
hydrolysis. Therefore, the rate-limiting step in SSF process for bioethanol production is
the enzymatic hydrolysis of the pretreated macroalgae cellulosic residue (MCR) into
glucose, rather than the fermentation of the reducing sugar to bioethanol by yeast.
Although the bioethanol concentration was found to generally increase with higher solid
loadings, but the percentage of theoretical yield was found to generally drop with higher
solid loadings. At the highest loading of 10% (w/v), the highest theoretical yield achieved
was only 31.1%. In fact, it was found that the lag phase (results not shown) for the
highest solid loading of 10% (w/v) reached almost 4 h. This lag phase is due to the
adaptation of the yeast to fermentation conditions and its duration is related with the solid
loading. The low theoretical yield at high solid loading could be due to insufficient
enzyme and furthermore the high initial sugar concentration at higher solid loading could
inhibit the enzyme leading to low cellulose hydrolysis. In addition, high solid loading can
also cause mixing problems due to higher viscosity of the mixture that lead to mixing
problem at the beginning of the SSF process. A similar result was also reported for
pretreatment of macroalgae by Lee et al. (2013). The substrate loading could not be
increased beyond the maximum of 6% due to high hygroscopic properties of macroalgae.
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Thus for this study, SSF process loaded with 4% (w/v) pretreated MCR and 45 FPU/g of
cellulase and 52 CBU/g of β-glucosidase was optimal for efficient production of
bioethanol. Interestingly, the bioethanol yield from pretreated MCR was found
comparable with the previously reported bioethanol yields from various macroalgae
materials such as brown algae by Lee and Lee (2012). In their study, a bioethanol
concentration of only 2.7 g/L was attained after 7 days of fermentation by using S.
cerevisiae (KCCM50550). In another paper by Kim et al. (2013) using S. cerevisiae as
the fermentation agent, a bioethanol yield of only 37.1% was attained after 24 h of SSF.
This shows that the highest bioethanol concentration (14.1 g/L) obtained from MCR
pretreated with Dowex (TM) Dr-G8 and followed by SSF process is much higher than
that reported by several other studies and can be obtained at a much shorter time. In
addition, this work utilized an environmental friendly solid acid pretreatment process
566 with mild temperature (120oC) and without the need of hazardous chemicals. (Lee,
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Li,(Lee, Ryu & Oh, 2013) (Lee & Lee, 2012) (Kim et al., 2013)
3.5. Scanning electron microscopy (SEM)
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Scanning electron microscopy (SEM) analysis revealed the morphological
features and surface characteristic of MCR pretreated with Dowex (TM) Dr-G8
compared with the untreated MCR. For the untreated MCR, there were highly ordered
fibrous structures of cellulose and the surface was continuous, even and smooth (Fig. 9A).
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After the solid acid pretreatment, the structure of pretreated MCR seems to have loosened
and resulting in exposure of internal structure and fibers (Fig. 9B). SEM image of the
pretreated MCR indicated that the pretreatment process resulted to the removal of
external fibers, which in turn increase surface area so that cellulose becomes more
accessible to enzymes. Increase in surface area and pore volume in pretreated solid
residues was reported to increase glucose yield during enzymatic hydrolysis of solid
residue (Ge, Wang & Mou, 2011).
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3.6 Porosity and surface analysis
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In general, the substrate surface area is a primary indicator of cellulose
accessibility. In this work, the Brunauer-Emmett-Teller (BET) surface area, pore volume,
and pore size of MCR before and after pretreatment were determined using nitrogen
adsorption. The values of surface area, pore volume, and pore size of the samples before
and after pretreatment were calculated and the results are listed in Table 3. Compared
with the untreated sample, the value of the surface area after the solid acid pretreatment
increased from 7.1728 to 9.3145 m2/g using the BET method. The cumulative pore
volume increased from 0.002618 to 0.007180 cm³/g, and the average pore size enlarged
from 11.2436 to 40.0427 Å after pretreatment. All of these values indicated that the
pretreatment using solid acid catalyst leads to a significant increase in the porosity of the
MCR, thereby resulting in the significant enhancement of enzyme accessibility toward
cellulose. (Guo & Catchmark, 2012).
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