Fuel 200 (2017) 380–386

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Full Length Article

Bioethanol production from acidic and enzymatic hydrolysates of mixed

microalgae culture
Hanieh Shokrkar, Sirous Ebrahimi ⇑, Mehdi Zamani
Biotechnology Research Centre, Faculty of Chemical Engineering, Sahand University of Technology, Tabriz, Iran

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

Reducing sugars extraction from Mixed microalgae culture

mixed culture of microalgae via
different pre-treatment strategies.

The effects of MgSO4 and CaCl2 as
lewis acid in acidic pretreatment on
sugar yield.

Comparison of the reducing sugar
yield of dried and wet microalgae
using enzymatic hydrolysis. Wet

Comparison of bioethanol yield of the Or

dried
fermentable sugars derived from OH
microalgae
different pre-treatment procedures. HO
O
OH
HO
OH

Acidic, alkaline, and enzymatic

Bioethanol Saccharomyces cerevisiae Glucose hydrolysis

a r t i c l e i n f o a b s t r a c t

Article history: Mixed microalgae cultures are considered as an attractive research area compared to traditional pure cul-
Received 25 July 2016 ture to dominate cultivation contamination risk and enhance economic feasibility of large-scale biofuel
Received in revised form 31 January 2017 production. However, pre-treatment and bioethanol production from mixed microalgae culture has not
Accepted 29 March 2017
been reported yet. Therefore, this study was aimed to evaluate the effect of different pre-treatment
strategies including acidic, alkaline, and enzymatic hydrolysis on the sugar extraction from mixed
microalgae. Besides, the effects of MgSO4 and CaCl2 as lewis acids in acidic pre-treatment on reducing
Keywords:
sugar yield were studied.
Acid pre-treatment
Bioethanol
Results showed that the mixture of dilute sulfuric acid and MgSO4 exhibited a higher sugar yield than
Enzymatic pre-treatment dilute acid. Among all pre-treatments used, the enzymatic treatment with thermostable enzymes showed
Lewis acid the highest recovery of 0.951 g extracted glucose/g total sugar. Moreover, the enzymatic pre-treatment of
Mixed microalgae wet microalgae was compared with dried ones at identical operational conditions and dried biomass con-
Thermostable enzymes centration of 50 g/l, similar sugar yields were achieved which would be advantageous to reduce the need
for drying of the microalgae biomass. Fermentation of the acidic and enzymatic treated samples to etha-
nol using Saccharomyces cerevisiae showed yield of 0.38 and 0.46 g/g glucose, corresponding to 76% and
92% of the theoretical values, respectively. The obtained results revealed that bioethanol yield after enzy-
matic hydrolysis of mixed microalgae culture are higher than those of acid hydrolysis.
Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction

⇑ Corresponding author. The quick growth of the world population and fast expansion of
E-mail address: sirous.ebrahimi@epfl.ch (S. Ebrahimi).
economies have both led to sharp increase in universal energy

http://dx.doi.org/10.1016/j.fuel.2017.03.090
0016-2361/Ó 2017 Elsevier Ltd. All rights reserved.
 H. Shokrkar et al. / Fuel 200 (2017) 380–386
consumption [1–3]. Therefore there is a strong incentive to reduce
the CO2 emissions and develop other energy sources as alternatives
to fossil fuels [4]. Algae would be good candidates for renewable
energy sources, receiving energy from the sunlight and building
their biomass by eliminating CO2 from atmosphere through photo-
synthesis [5].
Carbohydrates in microalgae biomass are mainly starch and cel-
lulose (with the absence of lignin), thus they are more easily
hydrolyzed to monosaccharide than other lignocellulosic materials
[6]. These carbohydrates in microalgae are not readily fermentable
to bioethanol, thus pre-treatment processes including chemical
(acid and alkaline) or enzymatic hydrolysis are crucial [7,8]. The
cost of pre-treatment contributes significantly to the total cost of
biomass conversion process, up to 30% [9]. Consequently, pre-
treatment has a great potential for improvement of converting bio-
mass to fermentable sugars.
To date, studies of ethanol production from sugars derived from
enzymatic hydrolysis of algae by thermostable enzymes are rare
[10]. The enzymatic hydrolysis using thermostable enzymes has
advantages in reducing the need for primary acid pre-treatment.
Despite the fact that there are few researches on bioethanol pro-
duction using pure culture micro- [6,11,12] and macroalgae [13–
17], bioethanol production from mixed culture of algae has not
been reported yet.
Cultivation of microalgae in pure culture is a main barrier for
large-scale biofuel production due to its costly aseptic process.
On the other hand, application of mixed culture of microalgae is
a desirable solution to dominate cultivation contamination risk,
operate in different conditions, and enhance economic feasibility.
Thus, mixed cultures are considered as an attractive research area
compared to traditional pure culture. Mooij et al. introduced the
concept of ‘‘survival of the fattest”, a strategy for enrichment of
species with a high storage compound productivity in mixed
microalgae culture [18]. Later on, Hassanpour et al. applied a gravi-
metric enrichment method for screening carbohydrate and lipid
accumulating species in a mixed microalgae culture [19].
In this work, sugars extraction from mixed culture of microal-
gae via different pre-treatment strategies include acid, alkaline,
enzymatic hydrolysis using thermostable enzymes were investi-
gated and compared. Subsequently, the bioethanol yield of the fer-
mentable sugars derived from different pre-treatment procedures
was compared through cultivating Saccharomyces cerevisiae yeast.
2. Materials and methods
2.1. Microalgae and growth medium
The original mixed culture of microalgae was obtained from a
freshwater area in Osku located in northwest of Iran (latitude is
37.91523 and longitude is 46.119901). The culture has been
enriched using the gravimetric method in sequence batch reactor
in our pervious study for starch storage [19]. This stock culture
sample was used to prepare pre-culture for 12 L photo-bioreactor.
Under the pre-culture condition, microalgae strains were grown
in a 1000 ml-Erlenmeyer flask with 500 ml working volume at
ambient temperature 25 1 °C, pH 8.9, constant light intensity
60 mmol m2 s1, and constant agitation rate of 150 rpm. The com-
position of medium was as follows (g/l): NaHCO3 (1.25); NaNO3
(0.8); KH2PO4 (0.2); MgSO4 (0.1); CaCl2 (0.1); KCl (0.1) and 2 ml/l
trace element solution containing (concentrations in mg/l): EDTA
(100); MnCl24H2O (10.12); FeSO47H2O (10); ZnSO47H2O (4.4);
(NH4)6Mo7O244H2O (3); CoCl26H2O (3.22); CuSO45H2O (3.14).
When optical density at 688 nm using UV/vis spectrophotome-
ter (Pharo 300, MERCK, Germany) reached 1.5, the grown algae
were transferred to the main photo-bioreactor.
381
2.2. Design and operation of photo-bioreactor
The photo-bioreactor (12 L) with working volume of 10 L was
inoculated with medium, which was pre-cultured in a 1000 ml-
Erlenmeyer flask. This photo-bioreactor was illuminated with an
external light source mounted on two sides of the photo-
bioreactor at a light intensity of approximately 260 mmol m2 s1
and worked at pH 8.9, 25 ± 1 °C and an aeration rate of 8 vvm.
The medium described above was used as nutrients in the photo-
bioreactor. The main photo-bioreactor was always inoculated with
the stock pre-culture in which the microbial contamination was
negligible. The regular microscopic observation using cell counting
by Neubauer counting chamber indicated that bacterial contami-
nation was always less than 2%, and no protozoa were observed.
The contamination risk was minimized through periodical renew-
ing the culture in the reactor with the stock pre-culture.
When the nitrogen source in the medium was completely con-
sumed, carbon dioxide was continuously fed with a rate of 0.1 vvm
into the microalgae culture to enhance intercellular carbohydrate
storage compounds, mainly as starch. End of the batch cultivation
(37 d), algae were harvested and used for enzymatic hydrolysis of
wet biomass. In addition, for the chemical and enzymatic hydroly-
sis experiments using dried biomass, the harvested algae were
dried at 80 °C for 24 h and ground into a powder.
2.3. Chemical hydrolysis
In chemical hydrolysis tests, the microalgae powders were
mixed separately with H2SO4 (0.5, 1, 2 M), HCl (0.5, 1, 2 M),
H3PO3 (0.5, 1, 2 M) and NaOH (0.5, 1, 2 M). In addition, the effects
of MgSO4 and CaCl2 as lewis acids in acidic pre-treatment on
reducing sugar yield were studied. The resulting slurries were then
autoclaved at 121 °C for 10, 20, 30, and 40 min. After hydrolysis,
the samples were allowed to reach room temperature. Then, sus-
pension was centrifuged at 4000g for 5 min and the supernatant
was taken for sugar content analysis. The chemical materials used
were based on the following references.
In the study conducted by Miranda et al. [20], the extraction of
sugars from the microalgae S. obliquus was investigated with
H2SO4, HCl and NaOH in autoclave at 121 °C for 30 min. Ho et al.
[6] pretreated microalgae C. vulgaris FSP-E with H2SO4 in autoclave
for 20 min. Nguyen et al. [21] used the hydrothermal acid pre-
treatment of microalgae Chlamydomonas reinhardtii with H2SO4
in an autoclave vessel at different temperatures (100, 110, and
120 °C) from 15 to 120 min. Zhou et al. investigated the hydrolysis
of algae chlorella for fermentable sugars in the presence of HCl and
MgCl2 at 180 °C from 10 min and 120 °C from 60 min [22].
2.4. Enzymatic hydrolysis
The enzymes used to hydrolyze carbohydrate components (cel-
lulose and starch) in the microalgae were purchased from alpha
enzyme company (Mashhad, Iran). They included thermostable
b-glucosidase/cellulase from Talaromyces emersonii, thermostable
a-amylase of Bacillus licheniformis (EC 3.2.1.1) and amyloglucosi-
dase from Aspergillus niger with activities of 1000 U/g,
145,000 TSAU/ml and 600 AGU/ml, respectively. A summary of
the optimal conditions for the enzymes is presented in Table 1.
For the enzymatic hydrolysis experiments, a sample with a con-
centration of 50 g/l dried microalgae powder was hydrolyzed in a
fixed volume of 20 ml containing citrate buffer with a pH value
of 5.5. Considering the optimum temperature for the enzymes
(table 1), the biomass was hydrolyzed for 3 h by b-glucosidase/
cellulase (denoted as enzyme 1), and a-amylase (denoted as
enzyme 2) at 65 and 95 °C, respectively. For the subsequent sac-
charification, temperature was reduced to 55 °C and amyloglucosi-
382 H. Shokrkar et al. / Fuel 200 (2017) 380–386

Table 1 Concentration of extracted reducing sugarðg=lÞ

Total reducing sugar yield% ¼
The thermostable enzymes used in the experiments and their optimal temperature Concentration of total carbohydrate in algaeðg=lÞ
and pH conditions.  100
Enzymes Microorganisms that Optimum Optimum ð1Þ
produced enzyme temperature (°C) pH
Furthermore, glucose yield was expressed as concentration of
b-Glucosidase/ Talaromyces Active at 4.0–6.5
extracted glucose per concentration of total carbohydrate in algae.
Cellulase emersonii temperatures up
to 80 °C Ethanol yield was calculated by the following equation:
a-Amylase Bacillus Licheniformis 95–115 °C 5.00–7.50
Amyloglucosidase Aspergillus Niger 20–70 °C 3.50–5.50 Concentration of ethanolðg=lÞ
Ethanol yield% ¼  100 ð2Þ
Concentration of glucoseðg=lÞ

dase (denoted as enzyme 3) was added and mixed for 3 h. The 3. Results and discussion
enzymatic hydrolysis was conducted at agitation rate of 400 rpm
and the total reducing sugar yield over time was measured. 3.1. Microalgae cultivation

2.5. Bioethanol fermentation Biomass and residual concentration of nitrogen in culture med-
ium were analysed for 37 days; the results are depicted in Fig. 1.
S. cerevisiae (ATCC 7921) was maintained in a solid medium As depicted in Fig. 1, after 29 days, the cell concentration
with composition as follows (concentrations in g/l): peptone (5); increased to 1.96 g/l whereas the nitrogen source (NO3) was com-
yeast extract (3); malt extract (3); glucose (10); and agar (20) at pletely depleted. Afterward, during the following eight days under
4 °C. Thereafter, pre-culture was prepared by transferring cells of nitrogen starvation and presence of CO2, microalgae accumulated
yeast from agar plate into a 250 ml-Erlenmeyer flask containing fixed carbon, mainly in the form of carbohydrates. Total carbohy-
100 ml of the culture medium containing (concentrations in g/l): drate content of microalgal biomass increased about 20.1% of VSS
glucose (100); KH2PO4 (3); MgSO4 (1); (NH4)2SO4 (1); Yeast extract in the absence of nitrogen. The observed increase is in agreement
(10) and 2 ml/l trace element solution.The Erlenmeyer flask was with the previous studies on pure culture of microalgae under
shaken in incubator at 150 rpm and 30 °C to grow the yeast aero- nitrogen starvation [26,27]. Also, the obtained results on day 37
bically for 24–48 h. The measurement of the optical density at showed that TSS, ash content, VSS, and total carbohydrate concen-
wavelength 600 nm (denoted as OD600) showed an exponential tration of microalgae were 2.05, 0.41, 1.64 and 0.596 g/l, respec-
growth behaviour. When OD600 reached 3 (mid log phase), a vol- tively. Therefore, the percentage of total carbohydrate was
ume of the inoculum (3% v/v) was centrifuged at 4000g for 5 min, calculated to be about 29% of TSS or 36% of VSS.
the supernatant was taken, and then yeast was added to the solu-
tion containing the hydrolysates of microalgae biomass. Acid
3.2. Hydrolysis of microalgae biomass
hydrolysates of microalgal biomass (50 g/l) using 0.5 M H2SO4
and 2.5% (w/v) MgSO4 by autoclaving at 121 °C after 40 min and
3.2.1. Chemical hydrolysis
also enzymatic hydrolysates of dried and wet biomass (50 g/l) after
Dilute acids decompose cellulose, and starch in the biomass to
9 h, were used for fermentation process. In addition, pH of the
release simple sugars, which can be fermented into bioethanol
microalgae hydrolysate was adjusted to 6.5 to provide a suitable
[28,29]. It has been reported that the hydrolysis kinetic depend
pH for ethanol production. The fermentation was performed anaer-
on the type of substrate, temperature, acid concentration, and reac-
obically at 30 °C shaken at 150 rpm.
tion time [29,30]. First, the effect of microalgae biomass concentra-
tion on reducing sugar and glucose yield via H2SO4 0.5 M and
2.6. Analytical methods
reaction time (10, 20, 30, and 40 min) by autoclaving at 121 °C
was investigated (Fig. 2).
For total suspended solids (TSS) determination, 40 ml of
When the microalgal biomass concentration was increased
microalgae culture was centrifuged at 4000g and washed with
from 25 to 50 g/l, the reducing sugar and glucose yield after
distilled water. Then supernatant was taken and the remaining
40 min remained constant at about 86 and 81%, respectively. How-
biomass was oven-dried at 100 °C for 24 h until the constant
weight was reached. Ash content was determined by igniting the
oven-dried sample at 550 °C for 1 h. Volatile suspended solids
amount (VSS) was computed by difference between TSS and ash
content. Total carbohydrate of microalgae biomass was deter-
mined by anthrone method [23]. Determination of residual con-
centration of nitrogen was performed using colorimetric method
according to Cataldo et al. (1975) [24]. Also, total reducing sugar
content of microalgae hydrolysates was determined with dinitros-
alicylic acid (DNS) method using the standard curve prepared with
glucose [25]. Glucose and ethanol concentrations were analyzed
using high-performance liquid chromatography (HPLC, RI detector)
with Eurokat H column. All experiments were performed in
duplicate.

2.7. Calculation of total reducing sugar, glucose and ethanol yield

Total reducing sugar yield was expressed as concentration of

extracted reducing sugar per concentration of total carbohydrate Fig. 1. Cell concentration and residual concentration of nitrogen in photo-biore-
in algae according to the following equation. actor (12 L) at pH 8.9, 25 ± 1 °C and an aeration rate of 8 vvm.
 H. Shokrkar et al. / Fuel 200 (2017) 380–386
ever, when the microalgae biomass concentration was increased
from 50 to 100 g/l, the reducing sugar and glucose yield decreased
from 86 and 81% to about 60 and 54%, respectively. Microalgae bio-
mass concentration is a significant factor, which can be optimized
to obtain the highest sugar yield. Therefore, the suitable microal-
gae biomass concentration was around 50 g/l. This biomass con-
centration was considered for the following experiments.
Moreover, the effect of hydrochloric acid (0.5, 1, 2 M) and sulfu-
ric acid concentration (0.5, 1, 2 M), 0.5 M H2SO4 and 2.5% (w/v)
MgSO4, 0.5 M H2SO4 and 2.5% (w/v) CaCl2 using 50 g/l of microal-
gae by autoclaving at 121 °C at different reaction times (10, 20,
30, 40 min) on the efficiency of microalgae hydrolysis was investi-
gated and the results are presented in Fig. 3.
As shown in Fig. 3, the increase in acid concentration improves
the sugar extraction yield. Thus, it can be concluded that higher acid
concentrations resulted in higher treatment rates [31]. Neverthe-
less, acid concentrations above 2 M, HCl 3 M and H2SO4 3 M, yielded
less reducing sugar about 82% and 84% after 40 min, probably due
to sugar degradation (data not shown). These results are in accor-
dance with previous study done by Miranda et al. [20]. Although
sulfuric acid is commonly used as acidic catalyst to convert many
feed stocks to fermentable sugars [32,33], it released less sugar than
hydrochloric acid. Although the underlying mechanism should be
further investigated, the obtained result is in agreement with Kim
et al. [34] and Israilides et al. [35]. Furthermore, Fig. 3 shows that
the highest reducing sugar yield was 94% using HCl 2 M or mixture
of H2SO4 0.5 M and 2.5% (w/v) MgSO4 for 30 min, while reducing
sugar yield when pre-treatment was performed using H2SO4
0.5 M for 30 min was obtained to be 72.18%. Moreover, hydrolysis
of microalgae biomass in the presence of MgSO4 and without
H2SO4 0.5 M yielded no more than 8.06% of total reducing sugar.
Some researchers have proposed magnesium sulphate (MgSO4)
and chloride salt (CaCl2) as lewis acid catalyze the hydrolysis of bio-
mass [36], although MgSO4 is a stronger lewis acid [37].
Afterwards, the effect of sodium hydroxide (0.5, 1, 2 M) and
phosphorous acid (0.5, 1, 2 M) on the efficiency of microalgae
hydrolysis with microalgae concentration of 50 g/l by autoclaving
at 121 °C for 20 and 40 min was investigated and the results are
presented in Fig. 4.
Fig. 2. Effect of microalgae biomass concentration on reducing sugar and glucose yield via sulfuric acid 0.5 M by autoclaving at 121 °C.
383
The result shows that sodium hydroxide released less sugar than
hydrochloric and sulphuric acid, probably due to sugar degradation
caused by the strong alkaline treatment. This result is in accordance
with the Miranda et al. work [20]. From Fig. 4, it can also be
observed that when algal biomass was pretreated with phospho-
rous acid (2 M) for 40 min, maximum yield of reducing sugar and
glucose reached about 48% and 42%. Nevertheless, these values
achieved about 80% and 75.5% in the treatment carried out with
NaOH (2 M). The similar results were observed by Kavitha et al.
[38], they achieved maximum glucose yield of 0.35 g/g algae and
0.055 g/g algae in the treatments carried out with 4% NaOH and
3% H3PO4 in autoclave at 121 °C for 40 min, respectively. Therefore,
it can be said that under the same concentrations and hydrolysis
time, H3PO3, a weak acid, released less reducing sugar and glucose
compared to its stronger counterparts, NaOH, HCl and H2SO4.
3.2.2. Enzymatic hydrolysis
3.2.2.1. Effect of enzyme composition on microalgae hydrolysis
yields. The enzymatic hydrolysis was conducted for 9 h and the
total reducing sugar yield over time was measured. Fig. 5 presents
compression of reducing sugar yields between enzymatic hydroly-
sis using 3 enzymes, and without adding enzyme 1.
As can be seen in Fig. 5, after reaction time of 3 h of adding b-
glucosidase/cellulase enzyme to hydrolyze the microalgae bio-
mass, the total reducing sugar yield reached about 33.26%. After-
ward, the addition of a-amylase and amyloglucosidase at time
6 h led to the total reducing sugar yield of around 93.64% and
97.06%, respectively. However, addition of a-amylase for 3 h with-
out b- glucosidase/cellulase showed a total reducing sugar yield of
around 61.19%, which afterward increased to 66.23% by addition of
amyloglucosidase. In addition, after reaction time of 9 h, glucose
yield using 3 enzymes, and without enzyme 1 was measured to
be 95.10% and 64.01%, respectively. It can be stated, when b-
glucosidase/cellulase was used to hydrolyze the microalgae bio-
mass, total reducing sugar, and glucose yield are much higher than
that of without b-glucosidase/cellulase.
As already mentioned, carbohydrates in microalgae biomass are
mainly cellulose and starch. Cellulose molecules are glucose poly-
mers linked together by b-1, 4 glucosidic bonds, as opposed to the
384 H. Shokrkar et al. / Fuel 200 (2017) 380–386

Fig. 3. Total reducing sugar and glucose yield extracted from microalgae biomass with hydrochloric and sulfuric acid by autoclaving at 121 °C.

Fig. 4. Total reducing sugar and glucose yield extracted from microalgae biomass with sodium hydroxide and phosphorous acid by autoclaving at 121 °C.

a-1, 4 and a-1, 6 glucosidic bonds for starch. In the enzymatic pre-
treatment of algae, b-glucosidase/cellulase hydrolyzed b-1, 4 glu-
cosidic bonds of algal cellulose. Whereas, a-amylase liquefied algal
starch to oligosaccharides through the hydrolysis of the a-1, 4 glu-
cosidic linkages, and then amyloglucosidase hydrolyzed a-1, 4 and
a-1, 6 glucosidic bonds of oligosaccharides into glucose. Therefore,
it is desirable to use three enzymes in the enzymatic pre-treatment
of microalgae, thus improving the hydrolysis yields even further.
3.2.2.2. Comparison of wet and dried microalgae. The hydrolysis of
wet microalgae without drying was also investigated under the
same conditions described earlier using Enzyme 1, Enzyme 2 and
Enzyme 3. Total reducing sugar yield obtained from the dried and
wet microalgae after enzymatic hydrolysis for 9 h was around
97.06% and 93.85%, respectively. As the results indicated, similar
efficiency was achieved without drying of microalgae biomass
revealing reduction of pre-treatment process costs. The price of
pre-treatment process is up to 30% of the total cost. Thus,
pre-treatment has a great potential for improvement of converting
biomass to fermentable sugars [9].The results obtained in this study
showed the sufficiency of a unique enzymatic hydrolysis step, with-
out the need for drying and primary acid pre-treatment step.
 H. Shokrkar et al. / Fuel 200 (2017) 380–386 385

Fig. 5. Effect of enzyme composition on microalgae hydrolysis yields.

Fig. 6. Glucose consumption and ethanol production from pretreated biomass of algae using: diluted sulfuric acid and MgSO4 treatment; enzyme treatment of dried biomass;
enzyme treatment of wet biomass.

3.3. Bioethanol production
Bioethanol production of hydrolysates of microalgal biomass
was investigated using yeast with separate hydrolysis and fermen-
tation (SHF) method. Yeast utilized the glucose and other nutrients
in the microalgae hydrolysates for bioethanol production. Glucose
consumption and ethanol production from acid hydrolysates of
microalgal biomass using 0.5 M H2SO4 and 2.5% (w/v) MgSO4 by
autoclaving after 40 min and also enzymatic hydrolysates of dried
and wet biomass for 9 h, are depicted in Fig. 6.
The obtained results showed that yeast could produce 6.41 and
6.01 g/l ethanol from 13.77 and 13.3 g/l of glucose derived from dried
and wet microalgae, respectively, using enzymatic hydrolysis after
24 h. The obtained yields were 0.46 and 0.45 g/g glucose, which were
92% and 90% of the theoretical values for dried and wet microalgae,
respectively. Therefore, the similar ethanol yield was obtained with-
out drying of microalgae revealing reduction of process costs.
Yeast could also produce 4.96 g/l ethanol from 13.05 g/l of glu-
cose derived from microalgae using acid hydrolysis (H2SO4 0.5 M
and 2.5% MgSO4 by autoclaving at 121 °C at 40 min) after 24 h.
386 H. Shokrkar et al. / Fuel 200 (2017) 380–386
The yield was calculated to be 0.38 g/g glucose, which was 76% of
the theoretical value. As the results indicate bioethanol yield after
enzymatic hydrolysis of algae are higher than those of acid hydrol-
ysis. The obtained results are in a good agreement with other pub-
lished papers [39–41]. However, in those studies, bioethanol
production from a pure culture of algae has been investigated in
most of which enzymatic hydrolysis needed a primary chemical
or physical pre-treatment. As a conclusion, enzymatic hydrolysis
would be exactly promising, due to higher yields, less corrosion
problems, lower utility consumption, and less inhibitory products
[10]. In addition, the size of inoculation in bioethanol production
is important in completing the fermentation process. Therefore,
the effect of yeast concentration on ethanol yield was also investi-
gated. It was found that when different inoculation volume of 3
and 10% v/v of S. cerevisiae culture were added to the fermentation
media containing the hydrolysates of microalgae, 13.77 g/l of glu-
cose, the batch time was reduced from 24 to 8 h. However, the
ethanol yields were independent of inoculation size and averaged
92% of theoretical.
4. Conclusion
This study demonstrated the feasibility of sugars extraction and
bioethanol production from mixed culture of microalgae. Among
all investigated pre-treatments for reducing sugar extraction, ther-
mostable enzymes including b-glucosidase/cellulase, a-amylase,
and amyloglucosidase could effectively hydrolyze microalgae bio-
mass for ethanol production. The results obtained in the study
showed that for sugar extraction, a unique hydrolysis step (enzy-
matic hydrolysis without the need for drying and primary acid
pre-treatment) is sufficient and therefore pre-treatment process
costs could be declined. After pre-treatment, glucose in acidic
and enzymatic hydrolysates of microalgae were converted into
ethanol using S. cerevisiae with the yield values of 0.38 and
0.46 g/g glucose, which were 76% and 92% of the theoretical ones,
respectively. Eventually, it can be stated that fermentation of the
glucose after enzymatic hydrolysis exhibited a higher bioethanol
yield than those of acid hydrolysis of microalgae.
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