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Bioresource Technology 201 (2016) 360364

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

Short Communication

Evaluation of different solvent mixtures in esterifiable lipids extraction


from microalgae Botryococcus braunii for biodiesel production
Pamela Hidalgo a, Gustavo Ciudad a,b, Rodrigo Navia a,b,c,
a
Scientific and Technological Bioresources Nucleus, Universidad de La Frontera, Casilla 54-D, Temuco, Chile
b
Department of Chemical Engineering, Universidad de La Frontera, Casilla 54-D, Temuco, Chile
c
Centre for Biotechnology & Bioengineering (CeBiB), Universidad de La Frontera, Casilla 54-D, Temuco, Chile

h i g h l i g h t s

 Different solvents and their mixtures were tested in microalgae lipid extraction.
 Total and esterifiable lipid extraction yield and efficiency was determined.
 Esterifiable lipid extraction yield of 19.2% was obtained with chloroformmethanol.
 This yield corresponds to a 98.9% esterifiable lipids extraction efficiency.

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

Article history: Non-polar and polar solvents as well as their mixtures were tested for the extraction of microalgae lipids
Received 2 October 2015 and thus, to evaluate their effect on total and esterifiable lipids extraction yields with potential to be con-
Received in revised form 9 November 2015 verted to biodiesel. The obtained results show an increase in lipids and esterifiable lipids extraction yields
Accepted 12 November 2015
when non-polar and polar solvent mixtures were used. The higher esterifiable lipids extraction yield was
Available online 19 November 2015
19.2% wt (based on dry biomass) using a chloroformmethanol mixture (75% v/v of methanol), corre-
sponding to a 98.9% wt esterifiable lipids extraction. In addition, esterifiable lipids extraction yield of
Keywords:
18.9% wt (based on dry biomass) was obtained when a petroleum ethermethanol mixture (75% v/v of
Lipid extraction
Microalgae
methanol) was used, corresponding to a 96.9% wt esterifiable lipids extraction.
Polar solvents 2015 Elsevier Ltd. All rights reserved.
Non-polar solvents

1. Introduction than switchgrass crops (Dismukes et al., 2008). Furthermore,


microalgae have a smaller ecological footprint because the land
Microalgae offer many potential advantages as a non-food feed- area needed for their cultivation is 12 orders of magnitude lower
stock for biodiesel production, as several microalgae species can than conventional crops (Widjaja et al., 2009). Conventional crops
accumulate high quantities of neutral lipids such as triglycerides such as soybeans, require approximately 20 million Ha for
and free fatty acids (FFA), containing between 2% and 40% lipids producing 1 quadrillion BTU, whereas microalgae culture only
by dry weight (Hidalgo et al., 2015a). Besides, microalgae present 200.000 Ha (Sheehan et al., 1998).
a fast growth and high productivity compared with agricultural Regarding lipids composition, it varies considerably from one
crops (Widjaja et al., 2009). Additionally, microalgae can be culti- specie to another, as some microalgae are richer in neutral lipids
vated in regions of low economic value, such as non-arable land compared to other species (Lv et al., 2010). Microalgae lipids usu-
or in a saline water medium, avoiding the competition for land ally comprise high levels of polar lipids (phospholipids and glycol-
destined to food production (Kanda et al., 2011). They can even ipids) and non-acylglycerol neutral lipids (such as hydrocarbons,
use less water than traditional oilseed crops, as according to sev- sterols, ketones, free fatty acids, carotenes and chlorophylls) and
eral authors, microalgae cultures require 8 times lower volumes in lower proportions acylglycerols. Acylglycerols are composed of
of water compared to rapeseed crops. However, microalgae water fatty acid esters bonded to a molecule of glycerol that acts as back-
consumption is similar to corn crops and up to 10 times higher bone, and are categorized according to the number of fatty acids
bonded to glycerol (mono-, di- and triglycerides). Microalgae fatty
Corresponding author at: Department of Chemical Engineering, Universidad de acids range from 12 to 22 carbons in length and can be either sat-
La Frontera, Casilla 54-D, Temuco, Chile. Tel.: +56 45 2325477; fax: +56 45 2732402. urated or unsaturated, which can be converted into biodiesel
E-mail address: rodrigo.navia@ufrontera.cl (R. Navia). (Halim et al., 2012).

http://dx.doi.org/10.1016/j.biortech.2015.11.031
0960-8524/ 2015 Elsevier Ltd. All rights reserved.
P. Hidalgo et al. / Bioresource Technology 201 (2016) 360364 361

The extraction of lipids from microalgae is an important step in the flask. The solvent is heated in the flask until boiling and the
the overall process of biodiesel production. The extraction of vapors rise to the condenser zone, condensing and dripping down
microalgae lipids is usually accomplished via solvent extraction contacting the sample.
methods, however, their use have been limited due to the high The solvent mixtures used to extract lipids from microalgae
energy demand of the oil extraction stage, added to low lipid were petroleum ether (with methanol or ethanol) and chloroform
extraction yields (Prommuak et al., 2012). In biodiesel production (with methanol or ethanol). The effect of different methanol and
using microalgae almost 90% of the process energy is consumed ethanol proportions added to the solvents was evaluated. The
during oil extraction. Therefore, any improvement in lipid extrac- proportions of methanol and ethanol incorporated in the solvent
tion yields will promote a significant impact on the economy of mixtures were 25% v/v, 50% v/v and 75% v/v. Besides, lipid extrac-
the overall process (Ehimen et al., 2010). tion with 100% v/v of methanol and ethanol was also evaluated.
Organic solvents are the normally used in lipid extraction pro- The solvent mixtures dosage used for lipids extraction was
cedures, however the mechanisms are not fully understood. 10 mL per g dry biomass. Extraction time was fixed at 5 h. All lipid
Halim et al. (2012) proposed a solventmicroalgal biomass interac- extraction experiments were performed in triplicate. After
tion mechanism in which the organic solvent penetrates through extraction, the solvents were removed by distillation in a rotary
the cell membrane into the cytoplasm and interacts with the neu- evaporator. The free solvent samples were weighed to gravimetri-
tral lipids by means of Van der Waals forces, forming an organic cally quantify the extracted lipids (ExL).
solventlipid complex. This organic solventlipid complex, driven
by a concentration gradient, diffuses across the cell membrane 2.4. Lipid extraction yield and efficiency
and as a result, the neutral lipids are extracted out of the cells
(Grima et al., 2013). In the case of microalgae, some neutral lipids Lipid extraction yield from dry biomass was calculated using Eq.
are in the cytoplasm also forming complexes with polar lipids. (1), where ExL represents the gravimetric quantification of lipids
These complexes are strongly linked via hydrogen bonds to pro- obtained from experimental data.
teins in the cell membrane. The use of non-polar organic solvents
ExLg
is inadequate to disrupt these membranelipidprotein associa- Lipid extraction yield % wt dry biomass :  100
tions, due to weak interactions with the complex. However, polar dry biomassg
organic solvents are able to break the lipidprotein associations 1
by forming hydrogen bonds with the polar lipids in the complex Lipid extraction efficiency was calculated using Eq. (2), where
(Halim et al., 2012). Additionally, non-polar/polar solvent mixtures TL corresponds to the gravimetric quantification of total lipids of
have been also used in the extraction of lipid complexes from B. braunii microalgae, using a methanol:chloroform mixture of
microalgae, trying to increase the total lipids extraction yield 2:1 v/v (Hidalgo et al., 2015b).
(Grima et al., 2013; Halim et al., 2012). However, the effect on
the esterifiable extractive fraction (i.e. the raw material for biodie- ExLg
Lipid extraction efficiency % wt of total lipids :  100
sel production) has been only little addressed. Therefore, the aim of TLg
this work was to study the effect of different solvents and solvent 2
mixtures on both the total and the esterifiable lipid fraction extrac-
tion yield and quality.
2.5. Esterifiable lipid extraction yield and efficiency

2. Methods The esterifiable lipid extraction yield from dry biomass was cal-
culated using Eq. (3), where EL represents the gravimetric quantifi-
2.1. Microalgal biomass cation using GC-FID of esterifiable lipid o lipids that are
transformed to fatty acid methyl esters (or biodiesel) obtained
Microalgae Botryococcus braunii race B (LB 572, UTEX) supplied from experimental data.
by Desert Bioenergy S.A. in Chile was used for lipid extraction
experiments. The biomass was dried in a tunnel dryer at 50 C ELg
Esterifiable lipid efficiency % wt dry biomass :  100
for 48 h until reaching 5% moisture. After this, the dried biomass dry biomassg
was milled and then fractioned using sieves (particle 3
size < 150 lm). Microalgae powder fractions were stored at 5 C
The lipid extraction efficiency was calculated using Eq. (4),
before use.
where TEL corresponds to the gravimetric quantification using
GC-FID of total esterifiable lipid of B. braunii microalgae.
2.2. Reagents
ELg
Esterifiable lipid efficiency % wt total esterifiable lipids :  100
TELg
Reagents and auxiliary materials used were boron trifluoride in
methanol (20% BF3 in MeOH) and butyl hydroxy toluene (BHT) 4
from Merck, potassium hydroxide (KOH), sodium chloride (NaCl),
sodium hydroxide (NaOH) and sodium sulfate (Na2SO4) were sup- 2.6. Esterifiable lipids quantification
plied by Emsure (Merck KGaA). The solvents methanol (MeOH, p.
a.), ethanol (EtOH, 96%), chloroform (CHCl3, p.a.), petroleum ether Esterifiable lipids (EL and TEL) correspond to free fatty acids
(boiling point in the range between 35 C and 60 C, p.a.) were sup- (FFA) as well as fatty acids from the neutral and polar lipids frac-
plied by Sigma Aldrich. tions. These lipids are suitable for esterification as in the reaction
an exchange of the fatty acid with the alkyl group of the alcohol
2.3. Experimental setup is produced, thus forming the esters. These compounds were deter-
mined by alkaline saponification and a subsequent esterification
Butt tube systems were used for lipids extraction. In this sys- into fatty acid methyl esters (FAME) according to AOCS method
tem, the sample is placed into folded filter paper. The filter paper (Ce 266) (AOCS, 2012). AOCS methods are the international stan-
with the sample is placed into the Butt tube and the solvent in dards for the analysis of the physicochemical properties of fats and
362 P. Hidalgo et al. / Bioresource Technology 201 (2016) 360364

oils. The quantification of esterifiable lipids was performed by gas-


eous chromatography (GC) coupled with flame ionization detector 40 A A
(FID). Methanol/petroleum ether

Lipid extraction Yield (% wt in dry biomass)


B B

Lipid extraction efficiency (% wt of total lipids)


Methanol/Chloroform 150
Ethanol/petroleum ether
2.7. Non-esterifiable lipids quantification 30 Ethanol/Chloroform
C
CD
Non-esterifiable fractions correspond to unsaponifiable lipids CDE DEFG
CDEF 100
(such as hydrocarbons, sterols, ketones and alcohols, among CDFEG CDE
20 DEFG EFGH
others) and non-lipidic fractions (such as proteins). The
FGHI
unsaponifiable lipids quantification was performed according to GHI
HI I HI I I
AOCS method (Cc-6a-53). Protein content in the extracted lipids
50
was determined using Kjeldahl method. 10

2.8. Chromatographic analysis


0
0% 25% 50% 75% 100%
A Clarus 600 chromatograph coupled to a flame ionization detec-
Solvent volume (methanol or ethanol; v/v)
tor (FID) from Perkin Elmer was used for identification and quantifi-
cation. A capillary column, Elite-5 ms (30 m  250 lm  0.1 lm) Fig. 1. Lipids extraction yield and efficiency using solvent mixtures with different
was used. The following temperature program was used: 150 C methanol or ethanol content. Data represents the mean values of two samples and
the error bars show the standard deviations. The different letters indicate a
for 3.5 min and then increasing temperature at a rate of 1.1 C/min
significant difference at p < 0.05.
up to 240 C. Both the injector and detector temperature were
250 C, and He was used as the carrier gas. The injection vials were
prepared by adding a 100 lL sample to 1000 lL of internal standard For a polar solvent content higher than 50% in the extractive
(C17:0, 1000 mg/mL). mixture, the lipids extraction process was conducted in homoge-
neous phase. Moreover, higher lipid extraction yields were
2.9. Statistical analysis observed when the polar solvent alone (methanol or ethanol)
was used in the extraction. As shown in Fig. 1, the extraction of
Variance analysis and Tukeys test were performed using JMP- lipids with methanol (100% v/v) was higher compared to the sol-
9software (SAS Institute Inc., Cary, NC). A p-value below 0.05 was vent mixtures due to the removal of polar membrane lipids and/
considered significant. or lipids associated to polar molecules. Maximum yields of 35.3%
wt (based on dry biomass) with methanol (100% v/v) and 30.0%
wt (based on dry biomass) with ethanol (100% v/v) were obtained.
3. Results and discussion The higher extraction of lipid with methanol is related to the high
polarity and dielectric constant of this solvent respect to ethanol
3.1. Lipids extraction with solvent mixtures (Mohsen-Nia and Amiri, 2013). In the extraction of lipids from
microalgae, the polarity of the organic solvent is critical. Conse-
High lipid extraction yields from microalgae using solvent mix- quently methanol was able to extract a higher content of structural
tures (polar and non-polar solvents) instead of using a single sol- lipids which are associated to polar lipids (phospholipids) and pro-
vents (non-polar) have been already reported in the literature teins, compared to ethanol (Hidalgo et al., 2014). However, these
(Halim et al., 2012). The extraction of lipids using a non-polar lipid extraction yields were higher compared to total extracted
organic solvent (petroleum ether, polarity index 0.1) showed a lipids (23.1% wt based on dry biomass) and therefore the lipid
low yield (Fig. 1). This result can be explained because the polar extraction efficiencies were higher than 100% as observed in
lipids fraction, mainly constituted by phospholipids and glycol- Fig. 1. These differences are related to the extraction of non-
ipids, cannot be extracted by this non-polar organic solvent. When lipidic compounds during the process.
a more polar organic solvent (chloroform, polarity index 4.1) was
used, a higher lipid extraction yield was obtained. Nevertheless,
high extraction yield of polar cellular constituents such as protein, 3.2. Esterifiable lipids extraction with solvent mixtures
pigments, carbohydrates, phospholipids and glycolipids, among
others, are also present in the extraction product. The solvent mixtures containing a polar and a non-polar solvent
According to Fig. 1, an increment of lipid extraction yield was were able to extract a higher amount of lipids. According to the
observed with the addition of a polar solvent in the extractive mix- results (see Fig. 1), an increase in the polarity of the extractive mix-
ture. The addition of a polar solvent increased the polarity of the ture produced a higher lipid extraction yield. In addition, the sol-
extractive mixture and the affinity to more polar lipids. This result vent mixtures containing a polar and a non-polar solvent were
indicates that lipids extraction is highly dependent on both sol- able to extract a higher amount of esterifiable lipids. The addition
vents and solvent ratios used in the mixture, contrary to the results of a polar solvent in the extraction mixture increased the solubility
obtained by Lee et al. (1996), who found that the solvent of more complex lipidic compounds and free lipids, which can be
mixture ratio presented no effect on lipid recovery. In addition, converted into biodiesel (Hidalgo et al., 2015c). The addition of a
Ryckebosch et al. (2012) reported the effect of solvent and solvent polar solvent such as methanol or ethanol until 75% v/v to the
mixtures in lipid extraction yield. They found that using a metha- extractive mixture increased the esterifiable lipids extraction yield
nolchloroform mixture the highest lipid extraction yield was and efficiency, as shown in Fig. 2. Depending on the polar and non-
obtained (23% wt of lipid extraction with 50% v/v of methanol), polar solvent characteristics, different esterifiable lipids extraction
even higher to that obtained with a lower methanol concentration yields were obtained. In fact, a maximum esterifiable lipids extrac-
in the methanolchloroform mixture (18% wt of lipid extraction tion yield of 19.2% wt (based on dry biomass) was obtained when
with 25% v/v of methanol) and a dichloromethaneethanol mix- using a chloroformmethanol mixture (corresponding to 98.9%
ture (12% wt of lipid extraction with 50% v/v of ethanol). wt esterifiable lipid extraction efficiency). In addition, an esterifi-
P. Hidalgo et al. / Bioresource Technology 201 (2016) 360364 363

25 70
A A
Methanol-petroleum ether

Estefifiable lipids efficiency (% wt of total esterifiable lipids)


B B
Methanol/petroleum ether Methanol-chloroform
60 Ethanol-petroleum ether
Methanol/chloroform Ethanol-chloroform
Estefifiable lipids yield (% wt in dry biomass)

20 A 100
Ethanol/petroleum ether A
Etanol/chloroform c
A
50

Content (% wt of the total lipids)


B B B
B
15 BC 75
40
B CD CD CDE
DE CDE
D
EF DE
10 FG 50 30

FG FG DE DE
G G
20 E E E
5 25

10

0
0% 25% 50% 75% 100% 0
Solvent volume (methanol or ethanol; v/v) 0% 50% 100%
Solvent volume (methanol or ethanol, % v/v)
Fig. 2. Esterifiable lipids extraction yield and efficiency using solvent mixtures with
different methanol or ethanol content. Data represents the mean values of two Fig. 4. Unsaponifiable lipids content in total lipids extract when using solvent
samples and the error bars show the standard deviations. The different letters mixtures with different methanol or ethanol content. Data represents the mean
indicate a significant difference at p < 0.05. values of two samples and the error bars show the standard deviations. The
different letters indicate a significant difference at p < 0.05.

25

A A 100

Methanol-petroleum ether Methanol-petroleum ether


20 Methanol-chloroform Methanol-chloroform
Unsaturated fatty acid content (% wt of total lipid )

Ethanol.-petroleum ether 80 Etanol-petroleum ether


A A
Ethanol-chloroform Etanol-chloroform
B
Content (% wt of total lipid)

C C
C
D
15 E
E E
60 F F

B B
10
C
40
D
E
5
F 20
G G
H H
0
0% 50% 100%
0
Solvent volume (methanol or ethanol, % v/v) 0% 50% 100%
Solvent volume (methanol or ethanol, % v/v)
Fig. 3. Protein content in the total lipids extract when using solvent mixtures with
different methanol or ethanol content. Data represents the mean values of two Fig. 5. Unsaturated fatty acid (UFA) content in total lipids extract when using
samples and the error bars show the standard deviations. The different letters solvent mixtures with different methanol or ethanol content. Data represents the
indicate a significant difference at p < 0.05. mean values of two samples and the error bars show the standard deviations. The
different letters indicate a significant difference at p < 0.05.

able lipids extraction yield of 18.9% wt was obtained when using a yield was reached when methanol was used in the extraction pro-
petroleum ethermethanol mixture (corresponding to 96.9% wt cess. This corresponds to 74% wt esterifiable lipid extraction effi-
esterifiable lipid extraction efficiency). Moreover, an esterifiable ciency. On the opposite, when ethanol was used, an esterifiable
lipids extraction yield of 14.9% wt was obtained when using a chlo- lipid extraction yield of 11.2% wt was obtained, corresponding to
roformethanol mixture (corresponding to 76.6% wt esterifiable an esterifiable lipid extraction efficiency of 57.4%.
lipid extraction efficiency). Finally, esterifiable lipids extraction Clearly, the total lipid extraction does not correlate with the
yield of 13.6% wt was obtained when using a petroleum ether esterifiable lipids extraction, as polar cellular constituents such
ethanol mixture (corresponding to 69.9% esterifiable lipid extrac- as protein and neutral lipids that do not contain fatty acid such
tion efficiency). Thus, it is clear stated that the esterifiable lipids as hydrocarbons, sterols and ketones (unsaponifiable lipids) are
extraction yield and efficiency is highly dependent on the type of also extracted (Figs. 3 and 4). An increment in the polarity of the
solvent or solvent mixtures used (Ryckebosch et al., 2012). extractive mixture could facilitate the extraction of more polar
Despite the high lipid extraction yield observed when a pure fractions such as polar membrane lipids or non-lipidic compounds
solvent was used, a decreasing esterifiable fraction (as shown in of polar nature. Instead, the reduction in polarity of the extractive
Fig. 2) was observed. This result is related to a solvent selectivity mixture could favor the extraction of non-polar neutral lipids
decrease, including in the extracted fraction non-lipidic com- mainly composed by non-acylglycerol lipids, i.e. unsaponifiable
pounds such as amino acids, carbohydrates and/or polar lipids that hydrocarbons, sterols and ketones as well as free fatty acids. In
do not contain fatty acids, such as carotenes and chlorophylls Fig. 3 is observed that the increase of polar solvent volume in the
(Azevedo et al., 2008). A 14.3% wt in esterifiable lipid extraction mixture produced a high protein extraction yield due to the
364 P. Hidalgo et al. / Bioresource Technology 201 (2016) 360364

polarity increment of the extractive mixture, producing a decrease Dismukes, G.C., Carrieri, D., Bennette, N., Ananyev, G.M., Posewitz, M.C., 2008.
Aquatic phototrophs: efficient alternatives to land-based crops for biofuels.
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