Bioresource Technology 201 (2016) 304311

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Experimental study and thermodynamic modeling for determining the

effect of non-polar solvent (hexane)/polar solvent (methanol) ratio and
moisture content on the lipid extraction efficiency from Chlorella vulgaris
Mohammad Malekzadeh a, Hamed Abedini Najafabadi a, Maziar Hakim a, Mehrzad Feilizadeh a,
Manouchehr Vossoughi a,b,, Davood Rashtchian a
a
Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran
b
Institute for Biotechnology and Environment (IBE), Sharif University of Technology, Tehran, 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

Lipid recovery was decreased for wet

biomass when solvent composed
mostly of hexane.

Fatty acid extraction performance
was decreased at the presence of
water.

Hexane/methanol (1:1 v/v) is the
most efficient solvent mixture for
lipid extraction.

Extraction performance was
improved by increase of the
temperature.

The UNIQUAC model can accurately
estimate the fatty acid recovery.

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

Article history: In this research, organic solvent composed of hexane and methanol was used for lipid extraction from dry
Received 3 November 2015 and wet biomass of Chlorella vulgaris. The results indicated that lipid and fatty acid extraction yield was
Received in revised form 23 November 2015 decreased by increasing the moisture content of biomass. However, the maximum extraction efficiency
Accepted 24 November 2015
was attained by applying equivolume mixture of hexane and methanol for both dry and wet biomass.
Available online 28 November 2015
Thermodynamic modeling was employed to estimate the effect of hexane/methanol ratio and moisture
content on fatty acid extraction yield. Hansen solubility parameter was used in adjusting the interaction
Keywords:
parameters of the model, which led to decrease the number of tuning parameters from 6 to 2. The results
Microalgae
Wet biomass
indicated that the model can accurately estimate the fatty acid recovery with average absolute deviation
Lipid extraction percentage (AAD%) of 13.90% and 15.00% for the two cases of using 6 and 2 adjustable parameters,
Organic solvent respectively.
Thermodynamic model 2015 Elsevier Ltd. All rights reserved.

1. Introduction of depletion of available fossil fuel reserves as well as negative

In recent decades, reliable alternative energy sources, such as
biodiesel have attracted wide spread attention, as a consequence
Corresponding author at: Department of Chemical and Petroleum Engineering,
Sharif University of Technology, Tehran, Iran. Tel.: +98 21 66164140; fax: +98 21
66005417.
E-mail address: vosoughi@sharif.edu (M. Vossoughi).
environmental effects of fossil fuel consumption (Shahid and
Jamal, 2008). Biodiesel enjoys many advantages, of which the most
important ones are the biodegradability (Kaercher et al., 2013), fea-
sibility to mix with fossil fuels (Demirbas, 2007), and non-toxicity
(Snchez-Arreola et al., 2015).
Production of biodiesel involves transesterification of triglyc-
erides found in plant and animal lipids (Savaliya and Dholakiya,

http://dx.doi.org/10.1016/j.biortech.2015.11.066
0960-8524/ 2015 Elsevier Ltd. All rights reserved.
 M. Malekzadeh et al. / Bioresource Technology 201 (2016) 304311
2015; Tarakowski et al., 2015). Recently, microalgae have gained
much attention as a promising resource for biodiesel production
due to substantial amounts of lipids (Mata et al., 2010), high
growth rate (Rawat et al., 2013), and cultivation capability in saline
water as well as nonagricultural land (Mackay et al., 2015).
Lipid extraction is a critical step in downstream processing of
biodiesel production from microalgae (Halim et al., 2012). Organic
solvent extraction is one of the most well-known processes used
for this purpose (Ramluckan et al., 2014). So far, various organic
solvents have been used for extraction of lipids from microalgae.
It has been shown by many researchers that the mixture of non-
polar and polar organic solvents facilitate the complete extraction
of all neutral lipids (Ramluckan et al., 2014; Sheng et al., 2011;
Wang et al., 2012). Nonetheless, further investigation is needed
on the choice of optimum ratio of non-polar/polar solvent mixture
to extract maximum amount of lipids.
To avoid high drying costs, the extraction technique should be
applied to wet biomass. However, the effect of the remaining water
in wet biomass on the lipid extraction performance is not well
known, yet and different scenarios have been proposed in this area,
so far. Most of the studies indicated that existence of moisture
decreases the mass transfer coefficient and consequently,
decreases the lipid extraction efficiency (Kanda et al., 2013; Liu
et al., 2013). On the other hand, some researchers reported that
moisture content has no effect on the extraction efficiency
(Halim et al., 2011) and some of them believe that existence of
water in the biomass would increase the extraction efficiency
due to the cell inflation (Medina et al., 1998). The effect of moisture
depends on several parameters such as algal strain, solvent type,
and the extraction temperature. Therefore, more investigation is
needed on this topic. Besides, it is also necessary to develop a
model to estimate the algal lipid extraction yield. There are some
examples for algal oil extraction modeling in literature (Halim
et al., 2011; Harrison et al., 2015; Liu et al., 2013). However, the
effect of moisture content and solvent composition is ignored in
these models.
Thermodynamic modeling is a suitable method to study the
behavior of equilibrium systems, such as extraction, and therefore
the effect of various parameters on the process can be analyzed by
using this method (Abedini Najafabadi et al., 2012; Hakim et al.,
2013; Nautiyal et al., 2014). In our previous work, we have used
this technique to model the purification process of extracted lipids
from microalgae (Abedini Najafabadi et al., 2015a).
In the present study, mixture of hexane (non-polar solvent) and
methanol (polar solvent) was used for one step oil extraction from
Chlorella vulgaris. The effect of various hexane/methanol ratios on
the recovery of lipids and fatty acids was examined. In order to
investigate the influence of biomass moisture content on the lipid
and fatty acid recovery yield, the extraction was also performed on
the wet biomass with moisture content of 70% and 85%. Further-
more, the extraction was carried out at five different temperatures
between 25 and 65 C. Finally, a thermodynamic model based on
solidliquid equilibrium was employed to estimate the fatty acid
recovery in the extraction process.
2. Methods
2.1. Materials
The solvents used in this work included hexane (99.9%, Merck)
and methanol (99.5%, Merck). Sulfuric acid (9598%, Merck) was
used as catalyst for transesterification reaction. All nutrients used
for culture media were laboratory or ACS grade. Methyl heptade-
canoate (99%, SigmaAldrich) was used as an internal standard
for fatty acid methyl ester quantification.
305
2.2. Microalgae strain and cultivation conditions strains
The C. vulgaris CCAP (211/19) was purchased from the Culture
Collection of Algae and Protozoa, Ambleside, Cumbria, UK. Microal-
gae was cultivated in a 5 l (ID, 16 cm; height, 25 cm) photobioreac-
tor containing sterilized Bolds Basal Media (BBM) (Stein, 1979) for
about two weeks, followed by one week of stressing in nitrogen-
starvation medium (N-BBM) as described by Abedini Najafabadi
et al. (2015b). The BBM media includes mostly phosphates such
as K2HPO4 (75 mg/L) and KH2PO4 (175 mg/L), nitrates such as
NaNO3 (250 mg/L), sulfates such as MgSO47H2O (75 mg/L), FeSO4
7H2O (4.98 mg/L), ZnSO47H2O (17.76 mg/L), and CuSO45H2O
(3.14 mg/L), as well as chlorides such as CaCl22H2O (25 mg/L),
NaCl (25 mg/L), MnCl24H2O (2.88 mg/L), and CoCl26H2O
(0.8 mg/L). Other materials such as KOH (31 mg/L), Na2EDTA
(50 mg/L), H3BO3 (11.42 mg/L), and (NH4)6Mo7O244H2O
(1.74 mg/L) are also used in this medium. 0.1 g/l/d of sodium acet-
ate was added to the medium to provide carbon source. The culture
was grown under 300400 lmol photon m2 s1 of artificial light
(continuous fluorescence illumination) and its temperature was
adjusted to 25 C. Rate of aeration was 1 vvm under an injection/
non-injection cycle of 1/3 min.
2.3. Harvesting and dewatering
For primary dehydration, the biomass was centrifuged at
4000 rpm for 10 min. A sample of the centrifuged algal biomass
was collected and lyophilized completely using freeze drier at
80 C. Weight of the dried sample was used to determine the
moisture content of the centrifuged biomass according to the fol-
lowing equation:
W wet  W dry
M %  100 1
W wet
where Wwet and Wdry are weight of wet and dried microalgae,
respectively.
Other wet biomass samples with different moisture contents
were prepared by adding specific amount of distilled water to
the centrifuged sample.
2.4. Extraction of microalgal crude oil
0.5 g of lyophilized algal biomass was mixed with 15 ml of sol-
vent in a flask. The sample was sonicated in an ice-bath at 38 kHz
for 20 min. The lipid extraction was performed by agitating the
mixture with a magnetic stirrer at 180 rpm and 25 C for 24 h. This
long time of extraction ensured the chemical equilibrium between
the solvent and algal biomass. For the oil extraction from the wet
microalgae, a given amount of wet biomass (equal to 0.5 g dry bio-
mass) was weighted, and the same procedure used for lyophilized
biomass was repeated. The resulting mixture was centrifuged at
4000 rpm for 10 min and the supernatant was collected.
2.5. Lipid and fatty acid determination
To determine crude lipids amount, the solvent was removed
using a rotary evaporator at the temperature of 50 C and the vac-
uum of 200 mmHg. After complete vaporization of the solvent, the
remaining lipids in the flask were weighed. The crude lipid recov-
ery from the algal biomass was calculated as follows:
W Lipid
RLipid %  100 2
W dry
where the RLipid and WLipid are the recovery and the weight of crude
lipid extracts, respectively.
306 M. Malekzadeh et al. / Bioresource Technology 201 (2016) 304311
The procedure of fatty acid determination was performed as
described in the literature by Lamers et al. (2010). In brief, the fatty
acids content of dried solvent extracts were converted to fatty acid
methyl esters (FAMEs), using a 3 ml solution of 5% (v/v) H2SO4 in
methanol. After extraction for 60 min with 3 ml hexane, 1 ll of
sample was analyzed by GCMS, essentially as described by
Hakim et al. (2013). The fatty acid recovery from the biomass
was calculated as follows:
W fa
Rfa %  100
W dry
where the Rfa and Wfa are the recovery and the weight of fatty acids
in the extraction solvent, respectively.
2.6. Thermodynamic modeling
According to the fundamental relations of thermodynamic, for
the extraction process at equilibrium the activity of each compo-
nent in raffinate (feed phase) and extract (solvent phase) must
be equal (Sharma, 2007):
aIi aIIi
thus:
wIi cIi wIIi cIIi
where wi and ci are mass fraction and activity coefficient of compo-
nent i in each phase, and superscripts I and II refer to the raffinate
and extract phases, respectively. Eq. (5) can be rearranged into a
relation of composition distribution between the phases:
wIIi cIi =cIIi wIi
A well-known thermodynamic model, the UNIQUAC model
(Abrams and Prausnitz, 1975), was used to express the activity
coefficients. This model is widely used to estimate the solid/liquid
phase equilibrium in various systems such as Mn(NO3)2H2O bin-
ary system (Arrad et al., 2015), D-glucose and sucrose in mixed sol-
vent mixtures (Peres and Macedo, 1997), synthetic paraffin
mixtures (Nasrifar and Fani Kheshty, 2011) and aqueous solutions
of sugars (Catt et al., 1994). UNIQUAC model has various interac-
tion parameters between each pair which is usually considered as
the adjustable parameters. It should be noted that in the present
study, the interaction parameters of hexanemethanol, metha-
nolwater, and hexanewater pairs were obtained from the litera-
ture (Iglesias et al., 2007). All of the fatty acids were considered as
one pair and the interaction parameters for pairs of fatty acid/hex-
ane, fatty acid/methanol and fatty acid/water (totally six parame-
ters) were obtained by minimizing the following objective
function:
X
N
2 crude lipids was increased by increasing the amount of polar sol-
X wcalc:
fa  wexpt:
fa
i1
where N is the number of experimental data points, subscript fa
stands for fatty acid and superscripts calc. and expt. refers to cal-
culated and experimental results.
In an innovative method, Hansen solubility parameter was used
to simplify the system and decrease the number of adjusting
parameters (Vetere, 2004). In this case, interaction parameters
are correlated to solubility parameters according to following
equation:
Dafa;i ajdfa  di j b
where Da are interaction parameters in the UNIQUAC model, and a
and b are new adjusting parameters for the UNIQUAC model. d is
the Hansen solubility parameter, the value of which is obtained
for different materials from the literature (Hansen, 2007) and is
reported in Table 1. It should be noted that by this assumption,
the number of adjusting parameters was decreased from 6 to 2.
The AAD (%) between the experimental data for fatty acid recov-
ery and those estimated by the thermodynamic model were calcu-
lated according to Eq. (9) (Press et al., 1987):
 
N Rexpt:  Rcalc: 
1X  fa;i fa;i 
AAD %    100 9
N i1  Rexpr:
fa;i

3
2.7. Statistical analysis
Each experiment was repeated at least three and at most four
times. Results were expressed as mean standard deviation.
GraphPad Prism 5 software was used for statistical analysis. A p
value <0.05 was considered statistically significant.
3. Results and discussion
3.1. Extraction of lipids from lyophilized biomass
4 In the present study, the main focus is on fatty acids rather than
total lipids, since the former is more expected for biodiesel produc-
tion. To select the best solvent mixtures, different equivolume mix-
5 tures of polar and non-polar solvents including hexane/methanol,
hexane/ethanol, hexane/1-propanol, chloroform/methanol, diethyl
ether/methanol, acetone/methanol and ethyl acetate/methanol
were studied and the results of crude lipid and fatty acid recovery
are shown in Fig. 1. As it can be observed, the solvent mixture of
chloroform/methanol has the highest amount of crude lipid and
6 fatty acid recovery. The extraction performance of hexane/metha-
nol is slightly lower than chloroform/methanol, but by considering
the high toxicity of chloroform, the former solvent mixture may be
a better option. Hexane/methanol has higher efficiency compare to
the mixture of hexane with other polar solvents such as ethanol
and 1-propanol. This is because of higher polarity of methanol
compare to ethanol and 1-propanol. Besides, hexane/methanol
has higher efficiently compare to the mixture of methanol with
other non-polar solvents such as diethyl ether, acetone, and ethyl
acetate. In the next step, the extraction efficiency of other ratios
of hexane/methanol was investigated to determine the optimal
solvent mixture.
Different portions of hexane and methanol, 0:1, 1:2, 1:1, 2:1,
and 1:0 (v/v), were used for lipid extraction from the lyophilized
algal biomass. The results of crude lipid and fatty acid recovery
are shown in Fig. 2, and the fatty acid profiles are reported in
Table 2. Pure non-polar solvent, hexane, had the least efficiency
in the extraction with 8.2 0.5 and 1.27 0.35 for the crude lipid
and fatty acid recovery, respectively. The extraction efficiency of
7 vent, methanol. The maximum fatty acid recovery of 2.30 0.53
was attained by using mixture of 1:1 (v/v) hexane and methanol.
The reason behind is that non-polar solvent is unable to remove
cell associated lipids and the high polar solvent cannot readily sol-
ubilize all the triacylglycerols (TAGs) (Abrams and Prausnitz,
1975). The maximum fatty acid/lipid ratio also belonged to hex-
ane/methanol 1:1, indicating the effectiveness of this solvent mix-
ture. The fatty acid composition for the described solvents is
shown in Fig. 3. As it can be observed, the composition of extracted
fatty acids was nearly the same for all the portions of hexane and
methanol.
8
3.2. Effect of moisture content on extraction yield
The effect of moisture content of the biomass on the lipid
extraction performance has also been considered. Extraction from
 M. Malekzadeh et al. / Bioresource Technology 201 (2016) 304311 307

Table 1
Adjustable parameters for the UNIQUAC model.

Obtained from Ref Iglesias et al. (2007) Obtained by minimizing Eq. (7) Obtained by using Eq. (8)
Da12 613.477 Da14 2397.28 a 33.118
Da13 5.326 Da24 764.436 b 5.455
Da23 503.86 Da34 2043.69
Da21 154.295 Da41 10.1895
Da31 542.081 Da42 392.515
Da32 755.464 Da43 726.28

1: hexane, 2: methanol, 3: water, 4: fatty acid.

Fig. 1. Crude lipid and fatty acid recovery from lyophilized biomass by using different equivolume mixtures of polar and non-polar solvents (each experiment was repeated
four times except the one using mixture of hexane and 1-propanol, which was repeated three times.)

the wet microalgae was performed using the same proportions of
non-polar and polar solvents which were already used for the lyo-
philized biomass. Crude lipid recovery for two different moisture
contents of 70 and 85% has been shown in Fig. 4. The crude lipids
extraction efficiency of pure hexane extremely decreased for wet
biomass. As it can be observed, the difference between crude lipid
recovery from the dry and wet biomass are statistically significant.
This can be explained by the limited contact between hydrophobic
hexane and biomass dispersed in the water phase. For hexane/
methanol 2:1, the crude lipid extraction was more efficient than
pure hexane. In the case of using this solvent mixture, the effi-
ciency of crude lipid extraction from the wet algal biomass was
lower than the lyophilized biomass. However, the decrease in
crude lipid content by changing from lyophilized to wet biomass
was lower for this solvent mixture in comparison with pure hex-
ane. This is due to the fact that methanol and water are miscible
in mixture of wet biomass and solvent mixture of hexane/metha-
nol 2:1. Decrease in the crude lipid extraction performance is more
intensive for the moisture content of 85%. For the biomass with
70% moisture content no significant difference was observed. How-
ever, crude lipids recovery from the biomass with 85% moisture
content is statistically significant in comparison with the lyophi-
lized biomass. When equivolume mixture of hexane and methanol
(1:1) was used, the difference between crude lipid recovery from
the lyophilized and wet biomass was not significant, indicating
the slight effect of moisture content on the extraction performance.
The same behavior was observed when solvents with higher ratio
of methanol (hexane/methanol (1:2) and (0:1)) were used. Hence,
the moisture content of the biomass did not affect the crude lipid
extraction efficiency in high concentration of methanol.
The results of fatty acid extraction from the lyophilized and wet
biomass with the moisture contents of 70% and 85% are compared
in Fig. 5, and the fatty acid composition are reported in Table 2. The
fatty acid recovery was decreased due to the presence of moisture
in the biomass; however, the profile of extracted fatty acids for dif-
ferent solvent mixtures was nearly the same as the dried microal-
gae. An extreme difference between fatty acid recovery from dry
and wet biomass was observed, using pure hexane as the solvent
because of weak efficiency of pure hexane in extracting crude.
The fatty acid extraction efficiency from the wet biomass was
decreased when other portions of methanol and hexane were used.
Moreover, difference in the fatty acid recovery was statistically sig-
nificant for most of the data points, due to the existence of water
which reduced TAG mass transfer rate into the extraction solvent.
Increase of the moisture content from 70% to 85% decreased the
fatty acid recovery in all of the data points; nevertheless, the
reduction was more intense when hexane was the dominant por-
tion of the solvent mixture. Similar to the lyophilized biomass,
the maximum fatty acids recovery was obtained by using hex-
ane/methanol (1:1) as the extraction solvent which was
308 M. Malekzadeh et al. / Bioresource Technology 201 (2016) 304311

Fig. 2. (A) Crude lipid and fatty acid recovery from lyophilized biomass and (B) fatty acid/crude lipid (%) using different mixtures of hexane and methanol (each experiment
was repeated three times except the one using equivolume mixture of hexane and methanol, which was repeated four times.)

Table 2
Lipid recovery, fatty acid recovery and fatty acid composition of extracted oil from lyophilized and wet algal biomass.

Test No. Hexane /methanol (v:v) RLipid (%) Rfa (%) Fatty acid composition (%)
C14:0 C16:0 C16:2 C18:0 C18:1 C18:2 C18:3 C20:0
Lyophilized biomass
1 1:0 8.20 0.50 1.27 0.35 5.3 2.2 29.5 3.2 0.6 0.4 0.3 0.2 1.9 0.3 11.9 3.6 47.0 2.5 3.6 0.5
2 2:1 9.80 1.22 1.88 0.44 6.3 2.8 30.2 7.6 1.1 0.3 0.4 0.1 1.9 0.2 8.9 2.5 47.3 2.3 4.0 0.8
3 1:1 10.20 0.40 2.30 0.53 7.2 1.9 39.4 1.0 1.0 0.4 0.4 0.2 2.1 0.2 4.9 0.7 40.9 2.2 4.2 0.1
4 1:2 10.00 0.95 2.12 0.10 7.0 0.8 34.8 0.6 1.2 0.1 0.4 0.1 2.1 0.2 7.4 1.0 42.8 0.5 4.2 0.1
5 0:1 11.30 0.50 1.77 0.31 7.8 1.5 33.8 4.3 1.2 0.1 0.2 0.1 2.4 0.1 6.7 2.4 43.8 2.3 4.0 0.6
Biomass with 70% moisture content
6 1:0 3.60 0.71 0.25 0.05 2.7 0.6 42.0 1.6 1.5 0.8 ND 5.1 1.4 9.2 1.4 33.9 3.0 5.7 2.0
7 2:1 8.70 0.14 0.72 0.06 7.5 3.8 34.7 6.4 1.5 0.7 0.3 0.1 1.1 0.1 5.3 1.3 41.5 1.7 8.1 1.5
8 1:1 10.00 0.28 2.02 0.33 6.7 1.2 38.0 1.9 1.1 0.4 0.3 0.2 1.8 0.2 7.1 2.6 39.0 0.9 6.0 1.7
9 1:2 11.50 0.14 1.46 0.24 8.1 0.6 40.2 4.5 1.4 0.2 0.6 0.1 1.8 0.1 5.2 0.3 38.9 1.3 3.8 0.7
10 0:1 10.90 0.42 1.40 0.04 9.7 1.0 40.1 0.3 1.2 0.3 0.4 0.1 2.4 0.5 5.3 0.1 36.8 1.7 4.0 0.4
Biomass with 85% moisture content
11 1:0 1.00 0.14 0.17 0.03 4.2 1.3 46.2 1.3 3.5 0.9 ND 4.2 1.3 4.1 1.4 32.5 3.4 8.2 1.3
12 2:1 6.70 0.14 0.39 0.11 8.5 0.6 39.6 6.2 1.5 0.8 0.4 0.1 1.8 0.3 2.2 1.3 40.2 5.9 8.7 0.7
13 1:1 10.40 0.85 0.95 0.16 6.0 2.8 39.3 3.8 0.6 0.9 0.6 0.1 1.4 0.4 4.8 0.8 35.9 2.0 7.2 2.9
14 1:2 10.70 0.37 0.86 0.27 8.4 1.6 36.4 0.2 1.8 0.8 0.4 0.1 2.2 0.5 5.0 0.3 34.8 5.6 7.1 2.4
15 0:1 10.80 0.55 0.84 0.30 8.5 1.4 39.5 7.9 1.1 0.5 1.0 0.2 3.4 1.7 6.8 1.3 36.9 2.0 5.8 1.0

2.2 0.33 and 0.95 0.16 for 70% and 85% moisture content. As it
can be observed in Table 2, the composition of extracted fatty acids
was nearly the same for wet and dried biomass.
3.3. Effect of temperature on extraction yield
In order to determinate the temperature effect, lipid extrac-
tion of the wet microalgae with the moisture content of 70%
was carried out at temperatures of 25, 35, 45, 55, and 65 C by
using equivolume mixture of hexane and methanol. Results of
crude lipids and fatty acids extraction are shown in Fig. 6. As it
can be seen, increasing the temperature led to the increase of
both crude lipid and fatty acid recovery. This behavior can be
attributed to the enhanced mass transfer kinetics at higher tem-
peratures. The increscent in the extraction performance was
more intense for higher temperature, in which the crude lipid
 M. Malekzadeh et al. / Bioresource Technology 201 (2016) 304311 309

Fig. 3. Composition of extracted fatty acids from lyophilized biomass by using various mixtures of hexane and methanol (each experiment was repeated three times except
the one using equivolume mixture of hexane and methanol, which was repeated four times.)

Fig. 4. The results of crude lipid extraction from lyophilized and wet biomass using different mixtures of hexane and methanol. = p < 0.05 versus dry conditions;
= p < 0.01 versus dry conditions; = p < 0.001 versus dry conditions. (Each experiment was repeated three times except the ones using equivolume mixture of hexane
and methanol for lyophilized and wet biomass, which were repeated four times.)

and fatty acid recovery were increased by about 17% and 36% at
65 C comparing to the 25 C.
3.4. Thermodynamic modeling
Lipid extraction may be affected by many factors such as sol-
vent type, biomass moisture content, and extraction temperature.
However, the experimental determination of fatty acid recovery
from biomass at different conditions may take a great deal of time
and energy. Thermodynamic modeling is a suitable method to esti-
mate the extraction process instead of performing experimental
tests.
The UNIQUAC model was used for estimation of activity coeffi-
cients during the extraction processes. Twelve of the experimental
data (test number 1, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15 in Table 2)
were randomly chosen for tuning the model parameters by mini-
mizing Eq. (7). The adjustable parameters of the proposed model
in two cases of using 6 and 2 adjusting parameters are reported
in Table 1. The interaction parameters between hexanemethanol,
methanolwater and hexanewater pairs, which were obtained
from the literature (Iglesias et al., 2007), are also presented in this
table. The remaining experimental data (test number 2, 8, 14 in
Table 2) were used to check the validity of the proposed thermody-
namic model. The results of fatty acid recovery estimation are
depicted in Fig. 5. As it can be seen, the proposed model can accu-
rately estimate the fatty acid recovery at different biomass mois-
ture contents and solvent mixtures. The average absolute
deviation (AAD%) to train the proposed model in two cases of using
6 and 2 adjustable parameters were 8.64% and 10.16%, respec-
tively. Besides, the average absolute deviation (AAD%) to validate
the model with 6 and 2 adjustable parameters were 13.90% and
15.00%, respectively. These results indicated the suitability of using
Hansen solubility parameters in calculating the interaction param-
eters of the UNIQUAC model, since this assumption decreased the
number of adjustable parameters to one-third, without a signifi-
cant decrease in the estimation efficiency. Besides, with respect
310 M. Malekzadeh et al. / Bioresource Technology 201 (2016) 304311

Fig. 5. Experimental and estimated data for fatty acid recovery from lyophilized and wet biomass using different mixtures of hexane and methanol. = p < 0.05 versus dry
conditions; = p < 0.01 versus dry conditions; = p < 0.001 versus dry conditions. (Each experiment was repeated three times except the ones using equivolume mixture
of hexane and methanol for lyophilized and wet biomass, which were repeated four times.)

Fig. 6. Effect of temperature on crude lipid and fatty acid recovery from wet microalgae using equivolume mixture of hexane and methanol. (Each experiment was repeated
three times except the ones at temperature of 25 and 35 C, which were repeated four times.)

to access to the limited number of experimental data, the model
with 2 adjustable parameters can lead to more reliable and reason-
able results. It should be noted that in this study, the thermody-
namic modeling has been used to estimate the extraction
efficiency of solvent mixture composed of hexane and methanol.
In the same way, this modeling procedure can be used to estimate
the fatty acid extraction efficiency by using other mixtures of polar
and non-polar solvents. However, the model parameters for new
solvents should be adjusted again by using experimental data.
The other approach is making a correlation to express the adjusta-
ble parameters in terms of the solvents physical properties such as
critical temperature, critical pressure and molecular weight. In this
case, the model can be used for a wide range of solvents. However,
this idea is out of the scope of this paper and is deferred to future
work.
4. Conclusion
In this study, the effect of solvent composition (hexane/metha-
nol) and biomass moisture content on algal lipid extraction was
investigated. Crude lipid extraction yield was increased by increas-
ing the methanol portion in the solvent mixture. However, the
maximum fatty acid recovery was achieved by using equivolume
mixture of methanol and hexane. Besides, increasing the moisture
content led to diminish the extraction performance. UNIQUAC
thermodynamic model was employed to investigate the influences
of moisture content and solvent ratio on the extraction efficiency.
The results demonstrated the accuracy of this model to predict the
fatty acid recovery yield during the extraction process.
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