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Fuel 200 (2017) 380–386

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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

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

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
sugar yield were studied.
Acid pre-treatment
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: (S. Ebrahimi).
economies have both led to sharp increase in universal energy
0016-2361/Ó 2017 Elsevier Ltd. All rights reserved.
H. Shokrkar et al. / Fuel 200 (2017) 380–386 381

consumption [1–3]. Therefore there is a strong incentive to reduce 2.2. Design and operation of photo-bioreactor
the CO2 emissions and develop other energy sources as alternatives
to fossil fuels [4]. Algae would be good candidates for renewable The photo-bioreactor (12 L) with working volume of 10 L was
energy sources, receiving energy from the sunlight and building inoculated with medium, which was pre-cultured in a 1000 ml-
their biomass by eliminating CO2 from atmosphere through photo- Erlenmeyer flask. This photo-bioreactor was illuminated with an
synthesis [5]. external light source mounted on two sides of the photo-
Carbohydrates in microalgae biomass are mainly starch and cel- bioreactor at a light intensity of approximately 260 mmol m2 s1
lulose (with the absence of lignin), thus they are more easily and worked at pH 8.9, 25 ± 1 °C and an aeration rate of 8 vvm.
hydrolyzed to monosaccharide than other lignocellulosic materials The medium described above was used as nutrients in the photo-
[6]. These carbohydrates in microalgae are not readily fermentable bioreactor. The main photo-bioreactor was always inoculated with
to bioethanol, thus pre-treatment processes including chemical the stock pre-culture in which the microbial contamination was
(acid and alkaline) or enzymatic hydrolysis are crucial [7,8]. The negligible. The regular microscopic observation using cell counting
cost of pre-treatment contributes significantly to the total cost of by Neubauer counting chamber indicated that bacterial contami-
biomass conversion process, up to 30% [9]. Consequently, pre- nation was always less than 2%, and no protozoa were observed.
treatment has a great potential for improvement of converting bio- The contamination risk was minimized through periodical renew-
mass to fermentable sugars. ing the culture in the reactor with the stock pre-culture.
To date, studies of ethanol production from sugars derived from When the nitrogen source in the medium was completely con-
enzymatic hydrolysis of algae by thermostable enzymes are rare sumed, carbon dioxide was continuously fed with a rate of 0.1 vvm
[10]. The enzymatic hydrolysis using thermostable enzymes has into the microalgae culture to enhance intercellular carbohydrate
advantages in reducing the need for primary acid pre-treatment. storage compounds, mainly as starch. End of the batch cultivation
Despite the fact that there are few researches on bioethanol pro- (37 d), algae were harvested and used for enzymatic hydrolysis of
duction using pure culture micro- [6,11,12] and macroalgae [13– wet biomass. In addition, for the chemical and enzymatic hydroly-
17], bioethanol production from mixed culture of algae has not sis experiments using dried biomass, the harvested algae were
been reported yet. dried at 80 °C for 24 h and ground into a powder.
Cultivation of microalgae in pure culture is a main barrier for
large-scale biofuel production due to its costly aseptic process. 2.3. Chemical hydrolysis
On the other hand, application of mixed culture of microalgae is
a desirable solution to dominate cultivation contamination risk, In chemical hydrolysis tests, the microalgae powders were
operate in different conditions, and enhance economic feasibility. mixed separately with H2SO4 (0.5, 1, 2 M), HCl (0.5, 1, 2 M),
Thus, mixed cultures are considered as an attractive research area H3PO3 (0.5, 1, 2 M) and NaOH (0.5, 1, 2 M). In addition, the effects
compared to traditional pure culture. Mooij et al. introduced the of MgSO4 and CaCl2 as lewis acids in acidic pre-treatment on
concept of ‘‘survival of the fattest”, a strategy for enrichment of reducing sugar yield were studied. The resulting slurries were then
species with a high storage compound productivity in mixed autoclaved at 121 °C for 10, 20, 30, and 40 min. After hydrolysis,
microalgae culture [18]. Later on, Hassanpour et al. applied a gravi- the samples were allowed to reach room temperature. Then, sus-
metric enrichment method for screening carbohydrate and lipid pension was centrifuged at 4000g for 5 min and the supernatant
accumulating species in a mixed microalgae culture [19]. was taken for sugar content analysis. The chemical materials used
In this work, sugars extraction from mixed culture of microal- were based on the following references.
gae via different pre-treatment strategies include acid, alkaline, In the study conducted by Miranda et al. [20], the extraction of
enzymatic hydrolysis using thermostable enzymes were investi- sugars from the microalgae S. obliquus was investigated with
gated and compared. Subsequently, the bioethanol yield of the fer- H2SO4, HCl and NaOH in autoclave at 121 °C for 30 min. Ho et al.
mentable sugars derived from different pre-treatment procedures [6] pretreated microalgae C. vulgaris FSP-E with H2SO4 in autoclave
was compared through cultivating Saccharomyces cerevisiae yeast. 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
2. Materials and methods 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
2.1. Microalgae and growth medium MgCl2 at 180 °C from 10 min and 120 °C from 60 min [22].

The original mixed culture of microalgae was obtained from a 2.4. Enzymatic hydrolysis
freshwater area in Osku located in northwest of Iran (latitude is
37.91523 and longitude is 46.119901). The culture has been The enzymes used to hydrolyze carbohydrate components (cel-
enriched using the gravimetric method in sequence batch reactor lulose and starch) in the microalgae were purchased from alpha
in our pervious study for starch storage [19]. This stock culture enzyme company (Mashhad, Iran). They included thermostable
sample was used to prepare pre-culture for 12 L photo-bioreactor. b-glucosidase/cellulase from Talaromyces emersonii, thermostable
Under the pre-culture condition, microalgae strains were grown a-amylase of Bacillus licheniformis (EC and amyloglucosi-
in a 1000 ml-Erlenmeyer flask with 500 ml working volume at dase from Aspergillus niger with activities of 1000 U/g,
ambient temperature 25 1 °C, pH 8.9, constant light intensity 145,000 TSAU/ml and 600 AGU/ml, respectively. A summary of
60 mmol m2 s1, and constant agitation rate of 150 rpm. The com- the optimal conditions for the enzymes is presented in Table 1.
position of medium was as follows (g/l): NaHCO3 (1.25); NaNO3 For the enzymatic hydrolysis experiments, a sample with a con-
(0.8); KH2PO4 (0.2); MgSO4 (0.1); CaCl2 (0.1); KCl (0.1) and 2 ml/l centration of 50 g/l dried microalgae powder was hydrolyzed in a
trace element solution containing (concentrations in mg/l): EDTA fixed volume of 20 ml containing citrate buffer with a pH value
(100); MnCl24H2O (10.12); FeSO47H2O (10); ZnSO47H2O (4.4); of 5.5. Considering the optimum temperature for the enzymes
(NH4)6Mo7O244H2O (3); CoCl26H2O (3.22); CuSO45H2O (3.14). (table 1), the biomass was hydrolyzed for 3 h by b-glucosidase/
When optical density at 688 nm using UV/vis spectrophotome- cellulase (denoted as enzyme 1), and a-amylase (denoted as
ter (Pharo 300, MERCK, Germany) reached 1.5, the grown algae enzyme 2) at 65 and 95 °C, respectively. For the subsequent sac-
were transferred to the main photo-bioreactor. 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

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) 380–386 383

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

Fig. 2. Effect of microalgae biomass concentration on reducing sugar and glucose yield via sulfuric acid 0.5 M by autoclaving at 121 °C.
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- same conditions described earlier using Enzyme 1, Enzyme 2 and
treatment of algae, b-glucosidase/cellulase hydrolyzed b-1, 4 glu- Enzyme 3. Total reducing sugar yield obtained from the dried and
cosidic bonds of algal cellulose. Whereas, a-amylase liquefied algal wet microalgae after enzymatic hydrolysis for 9 h was around
starch to oligosaccharides through the hydrolysis of the a-1, 4 glu- 97.06% and 93.85%, respectively. As the results indicated, similar
cosidic linkages, and then amyloglucosidase hydrolyzed a-1, 4 and efficiency was achieved without drying of microalgae biomass
a-1, 6 glucosidic bonds of oligosaccharides into glucose. Therefore, revealing reduction of pre-treatment process costs. The price of
it is desirable to use three enzymes in the enzymatic pre-treatment pre-treatment process is up to 30% of the total cost. Thus,
of microalgae, thus improving the hydrolysis yields even further. pre-treatment has a great potential for improvement of converting
biomass to fermentable sugars [9].The results obtained in this study Comparison of wet and dried microalgae. The hydrolysis of showed the sufficiency of a unique enzymatic hydrolysis step, with-
wet microalgae without drying was also investigated under the 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 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
Bioethanol production of hydrolysates of microalgal biomass and wet microalgae, respectively, using enzymatic hydrolysis after
was investigated using yeast with separate hydrolysis and fermen- 24 h. The obtained yields were 0.46 and 0.45 g/g glucose, which were
tation (SHF) method. Yeast utilized the glucose and other nutrients 92% and 90% of the theoretical values for dried and wet microalgae,
in the microalgae hydrolysates for bioethanol production. Glucose respectively. Therefore, the similar ethanol yield was obtained with-
consumption and ethanol production from acid hydrolysates of out drying of microalgae revealing reduction of process costs.
microalgal biomass using 0.5 M H2SO4 and 2.5% (w/v) MgSO4 by Yeast could also produce 4.96 g/l ethanol from 13.05 g/l of glu-
autoclaving after 40 min and also enzymatic hydrolysates of dried cose derived from microalgae using acid hydrolysis (H2SO4 0.5 M
and wet biomass for 9 h, are depicted in Fig. 6. 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

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