Bioresource Technology 243 (2017) 793799

Contents lists available at ScienceDirect

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

Surfactants assist in lipid extraction from wet Nannochloropsis sp

Chongchong Wu a,b,1, Ye Xiao a,1, Weiguo Lin b, Junying Zhu b, Hector De la Hoz Siegler a, Mingsheng Zong b,
Junfeng Rong b,
a
Department of Chemical and Petroleum Engineering, University of Calgary, T2N 1N4 Calgary, Alberta, Canada
b
Research Institute of Petroleum Processing, Sinopec, Beijing 100083, China

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

Surfactants were adopted to assist in

lipids extraction from algae.

Surfactants can completely replace
polar organic solvents in algal lipids
extraction.

Use of surfactants led to higher lipid
yields and larger amount of
saponifiables.

Lipid yield using algal-based
surfactants was similar to external
surfactants.

Lipid yield was correlated to dipole
moment, partition coefficient, and
polarity.

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

Article history: An efficient approach involving surfactant treatment, or the modification and utilization of surfactants
Received 13 May 2017 that naturally occur in algae (algal-based surfactants), was developed to assist in the extraction of lipids
Received in revised form 2 July 2017 from wet algae. Surfactants were found to be able to completely replace polar organic solvents in the
Accepted 4 July 2017
extraction process. The highest yield of algal lipids extracted by hexane and algal-based surfactants
Available online 6 July 2017
was 78.8%, followed by 78.2% for hexane and oligomeric surfactant extraction, whereas the lipid yield
extracted by hexane and ethanol was only 60.5%. In addition, the saponifiable lipids extracted by exploit-
Keywords:
ing algal-based surfactants and hexane, or adding oligomeric surfactant and hexane, accounted for 78.6%
Algae
Lipids
and 75.4% of total algal lipids, respectively, which was more than 10% higher than the lipids extracted by
Surfactants hexane and ethanol. This work presents a method to extract lipids from algae using only nonpolar organic
Algal-based surfactants solvents, while obtaining high lipid yields and high selectivity to saponifiables.
Polar organic solvents 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Biofuels are promising substitutes for fossil fuels given their
advantages of lower environmental impacts, economic diversifica-
tion, and the renewability of resources (Xiao et al., 2016). Microal-
gae is one of the most attractive feedstocks for biofuels production
Corresponding author.
E-mail address: rongjf.ripp@sinopec.com (J. Rong).
1
Chongchong Wu and Ye Xiao contributed equally to the job.
due to their high growth rates, high biomass yields, and low land
requirements (Daroch et al., 2013; Li et al., 2016). Among the dif-
ferent biofuels that can be produced from algae, biodiesel is one
of the most widely investigated biofuels (Hannon et al., 2010). It
is noteworthy that algae lipid extraction is a critical step in biodie-
sel production, making it necessary to develop efficient and cost-
effective approaches to extract algal lipids (Seo et al., 2015).
Extracting lipids from dry algae is relatively easier than from
wet algae; however, tremendous amounts of energy are needed
for drying algal biomass after harvesting (Dejoye Tanzi et al.,

http://dx.doi.org/10.1016/j.biortech.2017.07.010
0960-8524/ 2017 Elsevier Ltd. All rights reserved.
794 C. Wu et al. / Bioresource Technology 243 (2017) 793799
2013). Consequently, a promising solution to decrease energy and
cost for algal lipid extraction is to directly isolate lipids from wet al-
gae without a dewatering process (Samori et al., 2013).
However, current lipid extraction techniques from wet algae
rely on the use of large amounts of organic solvents (Chen et al.,
2012), especially mixtures of nonpolar and polar solvents (Sheng
et al., 2011). The use of these solvents causes serious environmen-
tal issues as well as increases operating costs (Huang et al., 2010).
Therefore, it is desirable to develop solvent-free techniques or at
least reduce the amount of solvents used to extract lipids from
wet algal biomass so that the algal biodiesel production process
will be safer, more environmentally friendly, and more economi-
cally competitive with traditional fuels (Ghasemi Naghdi et al.,
2016). Although demulsification is a potential method to eliminate
the requirement for organic solvents and separate algal lipids
directly, it has been demonstrated that algal emulsions are formed
after algal cell wall degradation, and the composition of algal
emulsions is rather complex, consisting of neutral lipids, polar
lipids, proteins, and other algal products, impeding the extraction
of neutral lipids with conventional methods (Akbarzadeh et al.,
2013; Aniket, 2012 b; Frhlich et al., 2015; Schwenzfeier et al.,
2013). Therefore, it is difficult to break algal emulsions and extract
the lipids directly.
Alternative approaches should be developed to decrease the
amount of organic solvents used for wet algal lipid extraction,
especially considering the high toxicity of some polar solvents,
which is indispensable for efficient extraction of lipids from wet al-
gae. It has been reported that surfactants, containing a hydropho-
bic tail and a hydrophilic head, could interact with algae cell
membranes, and assist in algae cell wall disruption (Ulloa et al.,
2012). By disrupting algae membranes, surfactants have been
reported to be a promising aid for algal lipid extraction (Lai et al.,
2016). Meanwhile, there are plenty of natural surfactants in algal
suspensions, such as monoacylglycerols, phospholipids, sophoroli-
pids, and lipopeptides (Dong et al., 2016). Although these surfac-
tants might not be effective in assisting in lipid extraction
directly, it is possible to change their properties, or produce other
algal surfactants through a saponification reaction to aid in lipid
extraction. If surfactants, especially algal-based surfactants prove
useful, then less organic solvents will be required to extract algal
lipids.
Although several techniques have been developed to extract
lipids from algae, few studies have reported on the use of surfac-
tants, or algal-based surfactants, to replace organic solvents to
assist in extracting algal lipids. This research aims to develop a
new technique to reduce the use of organic solvents in the lipid
extraction process from wet algal biomass by utilizing external
surfactants and algal-based surfactants to replace polar organic
solvents. The impact of reaction conditions including reaction time,
pH, and temperature were investigated. The research established
an efficient technique for lipid extraction from wet algae biomass
with lower operational costs and will contribute to the develop-
ment of algal biodiesel.
2. Materials and methods
2.1. Materials
The Nannochloropsis sp. used in this research was purchased
from Yantai Hairong Biology Technology Co., Ltd. (Yantai, China).
The total lipid content of the algae sample was 22.09 0.16%. Cel-
lulase (activity 2000 IU/g), acid protease (activity 2.0  105 IU/g),
neutral protease (activity 7.0  104 IU/g), alkaline protease (activ-
ity 2.0  105 IU/g), and pectinase (activity 2.0  104 IU/g) were
purchased from Beijing Hongrun Baoshun Technology Co. Ltd. (Bei-
jing, China). Lysozyme (activity 2.0  104 IU/g) was purchased
from Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China).
Analytical grade chloroform, petroleum ether (PE), hexane, metha-
nol, ethanol, dimethyl sulfoxide (DMSO), stearyl trimethyl ammo-
nium chloride (STAC), and sodium dodecyl sulfate (SDS) were
purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. (Bei-
jing, China). Non-ionic surfactant (NS) and oligomeric surfactant
(OS) were synthesized at the Research Institute of Petroleum Pro-
cessing (Beijing, China).
2.2. Total lipid content and lipid yield measurements
The total lipid content of algae was measured by the Folch
method after the algal cells were thoroughly homogenized (Wu
et al., 2017). For the lipid yield measurements, 10 mL of the sus-
pension after treatment was mixed with 20 mL of nonpolar organic
solvents (chloroform, hexane, or PE), and 10 mL polar organic sol-
vents (ethanol or methanol) for 30 min to extract lipids. The
obtained organic layer was then evaporated and dried in an oven
at 60 C for 1.5 h. The lipid yield was calculated according to Eq.
(1).
mass of extracted lipids
Lipid yield  100% 1
mass of total lipids
2.3. Algae treatment procedures
Algae cells were treated to disrupt the cell wall according to the
procedure that we developed previously (Wu et al., 2017). Briefly,
10 mL of Nannochloropsis sp. suspension (containing 3 g of Nan-
nochloropsis sp.) was adjusted to a pH of 9.5 by adding sodium
hydroxide solution (2 mM). The algal suspension was then trans-
ferred into a 30 mL stainless steel autoclave, and placed into a
rotary oven operating at 100 C for 8 h. After this pretreatment,
the pH of the algae suspension was adjusted to 5.0 using an acetic
acid solution (1 mM) before adding 200 IU/g each of cellulase, pro-
tease, lysozyme, and pectinase. This allowed the algal suspension
to be enzymatically treated at 50 C for 10 h.
2.4. Demulsification methods
After algae cell walls were degraded (from 2.3), the resulting
suspension went through one of the five demulsification processes,
and was subsequently centrifuged at 5000 rpm for 10 min to verify
if lipids had separated from the algal suspension after demulsifica-
tion. If there was distinct lipid layer, the lipid phase was separated.
In the thermal demulsification, the algal suspension was heated
in a rotary oven operating at 120 C, 140 C, or 160 C for 4 h. For
mechanical demulsification, the algal suspension was transferred
to centrifuge tubes and centrifuged at speeds ranging from 5000
to 12,000 rpm for 10 to 120 min. In the thermochemical demulsifi-
cation, the pH of the algal suspension was adjusted to either 2, 4, 6,
8, 10, or 12, and the algal suspension was then allowed to react in a
rotary oven operating at 120 C for 4 h. For the enzymatic demul-
sification, the pH of the algal suspension was adjusted to 5, 7, or
9.5 prior to treating with acid, neutral, or alkaline proteases, sepa-
rately (ranging from 100 IU/g to 1000 IU/g). The algae suspension
was then reacted in a rotary oven at 50 C for 4 h. Finally, in the
surfactant-assisted demulsification, varying amounts (60 mg/L,
100 mg/L, 150 mg/L, 300 mg/L, or 500 mg/L) of STAC, SDS, OS, or
NS were added into the algal suspension, and each of these treat-
ments were reacted at multiple temperatures (25 C, 35 C, and
50 C) for varying lengths of time (16 h) in a rotary oven.
 C. Wu et al. / Bioresource Technology 243 (2017) 793799 795

2.5. Surfactant-assisted extraction of algal lipids Table 1

Lipid yield of different combinations of organic solvents.

In parallel to demulsification method, surfactant-assisted Organic solvents Lipid yield Organic solvents Lipid yield
extraction of algal lipids techniques were explored. After the algal (%) (%)
cell walls were degraded (from 2.3), different types of external sur- Chloroform 78.0 DMSO + Chloroform 81.3
factants (STAC, SDS, OS, and NS) were added into the algal suspen- Chloroform 94.9 DMSO + Chloroform 95.5
sion. The algal suspension was then transferred into an autoclave + Methanol + Methanol
Chloroform 90.9 DMSO + Chloroform 92.2
and heated in a rotary oven at different temperatures (25 C, + Ethanol + Ethanol
35 C, 50 C, 70 C) for various time periods (16 h). After cooling, PE 26.3 DMSO + PE 30.5
lipids in the algal suspension were extracted using 20 mL of hex- PE + Methanol 50.6 DMSO + PE + Methanol 59.5
ane, and the lipid yield was measured according to Eq. (1) after PE + Ethanol 55.8 DMSO + PE + Ethanol 68.4
Hexane 30.2 DMSO + Hexane 35.9
evaporating hexane.
Hexane 51.2 DMSO + Hexane 65.3
+ Methanol + Methanol
Hexane + Ethanol 60.5 DMSO + Hexane + Ethanol 79.8
2.6. Algae-based surfactants assist in algal lipid extraction
Note: the margin of error was within 0.20% for all the experiments.
In addition to external surfactants, the utilization of algal-based
surfactants in aiding in lipid extraction was investigated. After
treatment (from 2.3), the algal suspension was adjusted to the Several properties of a solvent influence its ability to efficiently
alkaline pH range (813) by adding 2 mM sodium hydroxide solu- extract algal lipids, particularly dipole moment (D), partition coef-
tion. The algal suspension was put in a shaker at 200 rpm for car- ficient (log P), and polarity index (PI) (Sheng et al., 2011). Table 2
rying out the saponification reaction at different temperatures summarized D, log P, and PI for the solvent mixtures used in this
(50 C, 60 C, 70 C, 80 C, and 90 C) for 1 h. The pH of the algal research. Further investigation of the relationship between the
suspension was then adjusted using acetic acid to a pH of 5. After lipid yield and D, log P, and PI, showed linear trends with R2 of
that, lipids were extracted with 20 mL of hexane, and the lipid 0.93, 0.94, and 0.91, respectively. Because these properties also
yield was measured according to Eq. (1) after evaporating hexane. have relationship with each other, a principal component analysis
(PCA) was carried out. PCA can convert inter-correlated variables
into a new set of orthogonal variables (principal components).
2.7. Measurement of unsaponifiables According to the PCA results, the first principal component (PC1)
(Eq. (2)) accounted for 97.9% of the variation in the data. The value
The unsaponifiables in the obtained algal lipids were deter- of PC1 for each solvents combination was then calculated and lin-
mined according to the standard method ES ISO 18609 and fol- early fitted with the lipid yields to obtain Eq. (3). A higher linear
lowed the same steps developed by Huang et al. (2016). relationship with R2 of 0.94 was observed between lipid yield
and PC1 (Fig. 1). This result indicated that the higher dipole
moment, higher polarity index, and lower partition coefficient of
3. Results and discussion
the solvents resulted in a higher the lipid yield. The first principal
component is shown in Eq. (2), and the lipid yield could be pre-
3.1. Influence of organic solvents on lipid yield
dicted using Eq. (3) based on solvent properties.
Traditional solvent extraction methods involve the addition of PC1 0:580  D  0:574  log P 0:578  PI 2
large amounts of organic solvents. To investigate solvent-free
methods to extract algal lipids, several technologies, including Lipid Yield % 7:30  D  7:22  log P 7:27  PI 58:2 3
thermal, mechanical, thermochemical, and enzymatic methods
were investigated as the means to break the emulsions of dis-
rupted Nannochloropsis sp. algal cells. However, no lipid layer 3.2. Surfactant-assisted algae lipid extraction
was observed for any of these treatments. The formation of stable
algal emulsions (Fang et al., 2016) as well as the combination of It is clear that manipulating solvent combinations can increase
algal lipids with biomacromolecules (Cooney et al., 2009; Doan algal lipid yields, however, large amounts of organic solvents (sol-
and Obbard, 2011), such as proteins and polysaccharides, in the vents: algal suspension = 3:1, volume ratio) are still required. An
algal debris, could account for the ineffectiveness of the investi- alternative approach to reduce the usage of organic solvents is
gated demulsification methods. the use of surfactants to assist in algal lipids extraction. In the pre-
To compare lipid yield with surfactant-assisted extraction sent study, we found that adding surfactants and hexane increased
methods, solvent extraction methods were investigated. Different the algal lipid yield dramatically, compared with solvent extrac-
organic solvents, and their various combinations, were used to tion. In addition, a lower proportion of organic solvents were
extract lipids from the disrupted Nannochloropsis sp. algal suspen- employed (solvents: algal suspension = 2:1, volume ratio). To fur-
sion. As shown in Table 1, solvent mixtures of polar and nonpolar ther study the role of surfactants on algal lipid extraction, the influ-
organic solvents led to higher lipid yields compared with only non- ence of surfactant treatment on lipid yield prior to hexane
polar organic solvents. The highest lipid yield was obtained by extraction was investigated (Fig. 2).
using a combination of chloroform and methanol. It is worth not- As shown in Fig. 2, the lipid yield increased markedly after
ing that the lipid yields from hexane and petroleum ether extrac- treating with surfactants compared with hexane extraction. The
tion were much lower than using chloroform, though they are all lipid yield was 13.8% with hexane extraction, increasing to 23.0%,
classified as nonpolar solvents, which is consistent with their 42.7%, 49.6%, or 64.2% when 200 mg/L of SDS, STAC, NS, or OS
polarity. Interestingly, the addition of a small amount (2 mL) of was added to the algal suspension, respectively. The lipid yield
DMSO, a more polar solvent than methanol and ethanol, resulted when using OS surfactant and hexane (64.2%) was comparable to
in a substantial increase in the lipid yield by 0.6% to 31.9%. These the lipid yield of using a combination of hexane and ethanol
results indicate that the solvents properties play a critical role in (60.5%). This indicates that surfactants could substitute polar
the extraction of algal lipids. organic solvents to assist in the extraction of algal lipids.
796 C. Wu et al. / Bioresource Technology 243 (2017) 793799

Table 2
Solvent properties of mixed solvents used in this research.

Solvents Dipole moment (D) Log P Polarity

index
Chloroform 1.16 1.97 4.10
Chloroform + Methanol 1.31 1.07 4.43
Chloroform + Ethanol 1.29 1.21 4.47
PE 0.00 4.30 0.10
PE + Methanol 0.54 2.62 1.77
PE + Ethanol 0.52 2.77 1.80
Hexane 0.08 4.00 0.00
Hexane + Ethanol 0.57 2.57 1.73
Hexane + Methanol 0.59 2.42 1.70
DMSO + Chloroform 1.34 1.76 4.29
DMSO + Chloroform 1.48 0.92 4.61
+ Methanol
DMSO + Chloroform + Ethanol 1.46 1.05 4.64
DMSO + PE 0.26 3.95 0.54
DMSO + PE + Methanol 0.76 2.37 2.11
DMSO + PE + Ethanol 0.74 2.51 2.14
DMSO + Hexane 0.33 3.67 0.45
DMSO + Hexane + Ethanol 0.79 2.32 2.08
DMSO + Hexane + Methanol 0.81 2.18 2.04

Fig. 2. Influence of adding surfactants on lipid yield. All treatments were performed
in triplicate; error bars represent three sample standard deviations from the mean.

50 C with dosages of 200300 mg/L for 2 h. This lipid yield was

Fig. 1. Relationship between lipid yield and solvent properties. PC1 is the first
principal component from PCA analysis of the solvent properties,
PC1 0:580  D  0:574  log P 0:578  PI; D is the dipole moment, P is the
partition coefficient, and PI is the polarity index.
In order to further improve the lipid yield extracted by OS sur-
factants and hexane, the influence of surfactant treatment condi-
tions were investigated (Fig. 3). Lipid yield increased with
increased temperature and reached the highest value (78.0%) when
the temperature was 50 C (Fig. 3a). Further increasing the temper-
ature to 70 C led to a decrease in lipid yield (54.9%). On the other
hand, the lipid yield increased from 68.1% to 78.1% by increasing
the reaction time from 1 to 2 h; however, it reduced gradually to
75.1% for 6 h of treatment (Fig. 3b). Meanwhile, the increase in sur-
factant dosage resulted in an enhancement in lipid yield initially,
as the lipid yield increased from 67.0% to 78.1% when the surfac-
tant dosage went from 100 mg/L to 200 mg/L (Fig. 3c). However,
the lipid yield did not change substantially when the dosage
increased from 200 mg/L to 300 mg/L, and further increasing sur-
factant dosage led to a notable decrease in the lipid yield. The lipid
yield peaked (78.2%) when surfactant treatment conditions were at
much higher than that reported by Lai et al. (2016) for Scenedesmus
wet biomass, which was less than 30% even though surfactants
were used to assist in wet algae cell wall disruption. Lai et al.
(2017) also employed surfactants to assist in extracting lipids from
Chlorella biomass and achieved similar lipid recovery efficiencies as
the present study. However, higher amounts of surfactants (7700
9113 mg/L, transferred from mole concentration according to the
molar weight of the surfactants) were added and larger volumes
of more polar solvents (ethyl acetate: algal suspension = 3:1) were
applied in that study (Lai et al., 2017). Meanwhile, compared to a
pure solvent extraction, the lipid yield after addition of surfactants
in the present study was similar to that of extracting using a com-
bination of DMSO + hexane + ethanol (Table 1). Therefore, with the
surfactant treatment, the solvent required for extracting algal
lipids decreased substantially, and the use of polar solvents was
completely avoided.
Polar lipids and proteins in the algal oil enhance the emulsifica-
tion of algal neutral lipids, making them difficult to extract (Aniket,
2012a). Neutral and nonpolar lipids can combine with each other,
or with proteins, through hydrophobic or van der Waals interac-
tions, and polar lipids are connected to proteins by hydrogen bond-
ing and electrostatic associations (Grima et al., 2013). Organic
solvents need to penetrate the biomass in order to obtain a high
lipid yield (Mercer and Armenta, 2011). Although hexane can help
extract neutral lipids from algae, the hydrogen bonds and the van
der Waals interactions make it difficult to extract lipids using only
nonpolar organic solvents (Hussain et al., 2015). By adding surfac-
tants, they can bind with the hydrophobic parts of the cell debris,
therefore, hydrophobic or van der Waals interactions between
lipids and proteins are disrupted, and lipids can be released (Lai
et al., 2016). The algal cell debris is negatively charged due to the
presence of certain groups such as carboxyl, hydroxyl, and phos-
phates (Huang and Kim, 2013). Therefore, compared with anionic
SDS, the cationic STAC leads to higher lipid yield through interact-
ing with negatively charged groups. The hydrophilic group of OS is
more positively charged than STA, hence, OS can interact with neg-
atively charged cell debris through electrostatic interactions and
hydrophobic interactions to release algal lipids. This could help
explain the highest lipid yields when using OS compared with
other surfactants. The hydrogen bonding interactions between
 C. Wu et al. / Bioresource Technology 243 (2017) 793799
Fig. 3. Influence of surfactant reaction conditions on lipid yield. (a) Effect of temperature, 200 mg/L surfactants dosage for 4 h; (b) Effect of reaction time, 200 mg/L
surfactants dosage at 50 C; (c) Effect of surfactants dosage, reacting at 50 C for 2 h. All treatments were performed in triplicate; error bars represent three sample standard
deviations from the mean.
eleven oxygen atoms in NS and hydrogen atoms in protein could
possibly account for the high lipid yield when adding NS.
Altering extraction conditions can influence lipid yield when
using surfactants to assist in lipid extraction, the increase in tem-
perature, reaction time, and surfactant dosage all had a similar
effect on the lipid yield, which first increased and then decreased.
This trend implies that there is an optimum condition when using
surfactants for algal lipid extraction. After algal cell wall degrada-
tion, phospholipids can still rearrange to form a bimolecular layer,
or liposomes, which are similar to cell membranes and can encap-
sulate lipids droplets that are combined with cell debris. Therefore,
it is still difficult to extract lipids from algal suspensions by using
only nonpolar solvents, and polar solvents are still required to
obtain higher lipid yields. However, appropriate surfactants can
help disrupt the phospholipid liposomes through transmembrane
lipid motion, leakage, and vesicle lysis and reassembly
(Ahyayauch et al., 2010; Lesieur et al., 2003). The phospholipids
can form mixed micelles with the external added surfactants that
results in the solubilisation of the liposomes (Lichtenberg et al.,
2013), which leads to the release of the substances contained
within the liposomes. The released lipids droplets and the phos-
pholipids could then be readily extracted by nonpolar solvents.
The micelle formation process is typically endothermic at low tem-
peratures and exothermic when temperature is high (Opatowski
et al., 2002). As a result, the formation of mixed micelle of surfac-
tants was enhanced with the increasing of temperature initially,
which led to the solubilisation of the liposomes and increased
the lipid yield. In addition, higher temperature could also promote
mass transfer process, leading to higher lipid yield. However, fur-
ther increase of the temperature above the optimum point inhib-
797
ited the formation of the micelle and lowered the lipid yield. By
extending reaction time, and adding more surfactants, the solubil-
isation of the liposomes was enhanced. Therefore, longer time, and
more surfactants initially led to an increase in the lipid yield. On
the other hand, further increase in reaction time, and surfactant
amount would produce more mixed micelles. The existence of
these micelles can increase the solubility of nonpolar solvents as
well as some neutral lipids in the water (Shinoda and Kunieda,
1973). In this scenario, some nonpolar solvents and lipids will
remain in the water phase, resulting in a decrease in the lipid yield
after the optimum conditions are reached.
3.3. In situ algae-based surfactants for algal lipids extraction
As discussed above, the addition of surfactants could increase
the algal lipid yield with hexane extraction. Although there are sur-
factants in the original Nannochloropsis sp., they were not useful to
assist in lipid extraction in their natural form. Bio-surfactants can
bind with each other and with neutral lipids through hydrophobic
interactions, leaving the hydrophilic head exposed to the outside to
form micelles or liposomes (Dong et al., 2016). These stable emul-
sions caused by naturally occurring bio-surfactants make it diffi-
cult to extract algal lipids by nonpolar organic compounds. In
this section, instead of adding external surfactants, we produced
algal-based surfactants in situ to assist in the hexane extraction
of lipids. The saponification reaction was employed to alter the
properties of bio-surfactants naturally present in the algal suspen-
sion, and their effects on the lipid yield was further investigated
(Fig. 4).
798 C. Wu et al. / Bioresource Technology 243 (2017) 793799
Fig. 4. Influence of saponification reaction on lipid yield. (a) Effect of alkaline pH, reaction temperature 80 C; (b) Effect of react temperature, alkaline pH 12.0. All treatments
were performed in triplicate; error bars represent three sample standard deviations from the mean.
As shown in Fig. 4, compared to the lipid yield of 30.2% when
only hexane was used, the lipid yield increased substantialy to
58.9% after the saponification reaction was performed at pH 8.0.
The lipid yield was further enhanced with an increase in pH, but
was followed by a decrease when the pH exceeded 12. On the other
hand, increasing temperature from 50 C to 70 C resulted in an
obvious increase in lipid yield from 66.6% to 78.6%. However, fur-
ther increases in the temperature over 70 C led to a decrease in
lipid yield. After optimizing the saponification reaction conditions,
the maximum lipid yield was 78.6%, which was 48.2% higher than
only using hexane and 18.1% higher than using hexane and etha-
nol. Moreover, the lipid yield was essentially the same compared
to using a DMSO, hexane, and ethanol mixture. These results indi-
cate that the saponification of naturally occurring algal surfactants
can replace polar organic solvents to help extract algal lipids,
thereby reducing the need for organic solvents.
During the saponification reaction, lipids are transformed to
fatty acid salts and glycerin or other alcohols under alkaline condi-
tions (Schuchardt et al., 1998). The produced fatty acid salts have a
high hydrophile-lipophile balance (HLB) value and low packing
parameter (Yan et al., 2007), which is similar to the external added
surfactants for the solubilisation of liposomes (Lichtenberg et al.,
2013). Consequently, the fatty acid salts generated from the
saponification reaction will act in a similar way as the externally
added surfactants by breaking the interactions between lipids dro-
plets and cell debris. In addition, the alkaline hydrolysis of the
phospholipids could also assist in the disruption of the bilayer to
release lipids. Therefore, the lipid yield increased with the increase
in saponification pH and temperature initially. However, further
hydrolysis of the lipids might lead to a decrease in lipid yield
due to the loss of glycerin and other alcohols when temperature
and pH exceeded optimum conditions.
3.4. Analysis of algal lipids
During the lipid extraction process, non-lipid fractions are also
extracted. The content and composition of lipids extracted from
algae may vary depending on the extraction method (DOca et al.,
2011). Therefore, in addition to lipid yield, selection of the most
appropriate lipid extraction method should also consider the selec-
tivity towards the desired lipid fractions (Pragya et al., 2013).
Saponifiable lipids are required for algal biodiesel production, as
a high saponifiable lipid content is preferred (Urrutia et al.,
2016). Saponifiables and unsaponifiables of algal lipids using dif-
ferent methods were determined in order to choose the most
appropriate extraction technique.
Table 3
Saponifiables and unsaponifiables of algal lipids.
Organic compounds Saponifiable lipid Unsaponifiable lipid
(%) (%)
Chloroform + Methanol 40.4 59.6
Hexane + Ethanol 65.5 34.5
DMSO + Hexane + Ethanol 64.9 39.1
Surfactants + Hexane 75.4 24.6
Algal-based surfactants + Hexane 78.6 21.4
As shown in Table 3, the content of saponifiables was the high-
est when the algal-based surfactants and hexane were used in
combination to extract lipids. Lipid yield using this method was
13.0% higher than using hexane and ethanol, and 38.2% higher than
using chloroform and methanol, indicating that the combination of
the algal-based surfactants and hexane extraction is the most suit-
able method for the extraction of algal lipids for biodiesel produc-
tion. The saponifiables content of external surfactants and hexane
ranked second among the five extraction methods, and was only
3.2% lower than the algal-based surfactant and hexane combina-
tion, and 9.9% higher than hexane and ethanol method. The results
indicated that the in situ saponification of algal surfactants not
only improved the lipid yield, but also increased the saponifiable
content, all the while decreasing the volume of organic solvents
used in the extraction process. The reduction of organic solvents
significantly minimize the environmental impacts of algal biodie-
sel as well as its cost.
4. Conclusions
An innovative approach was developed to extract algal lipids
effectively. External and algal-based surfactants proved to be effi-
cient replacements of polar solvents to improve lipid extraction
and obtain saponifiables, laying the foundation for producing algal
biodiesel with a reduced use of organic solvents. Using oligomeric
surfactants and hexane resulted in a lipid yield as high as 78.8%
which is approximately 18% higher than using hexane and ethanol.
Similar results were achieved with algal-based surfactants. More-
over, the saponifiables of algae lipids extracted by these two meth-
ods were 78.6% and 75.4%, respectively, much higher than using
hexane and ethanol.
Acknowledgements
The project was funded by Research Institute of Petroleum Pro-
cessing Sinopec (S215104). The authors acknowledge Kady Lyons
 C. Wu et al. / Bioresource Technology 243 (2017) 793799
from University of Calgary for manuscript revision. Chongchong
Wu and Ye Xiao are grateful for the scholarship from Department
of Chemical and Petroleum Engineering, University of Calgary.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2017.07.
010.
References
Ahyayauch, H., Bennouna, M., Alonso, A., Goi, F.M., 2010. Detergent effects on
membranes at subsolubilizing concentrations: transmembrane lipid motion,
bilayer permeabilization, and vesicle lysis/reassembly are independent
phenomena. Langmuir 26, 73077313.
Akbarzadeh, A., Rezaei-Sadabady, R., Davaran, S., Joo, S.W., Zarghami, N.,
Hanifehpour, Y., Samiei, M., Kouhi, M., Nejati-Koshki, K., 2013. Liposome:
classification, preparation, and applications. Nanoscale Res. Lett. 8, 102.
Aniket, K. 2012a. Methods of and systems for dewatering algae and recycling water
therefrom, US Patents 8182689 B2.
Aniket, K. 2012b. Methods of and systems for isolating carotenoids and omega-3
rich oil products from algae, US Patents 8153137 B2.
Chen, M., Liu, T., Chen, X., Chen, L., Zhang, W., Wang, J., Gao, L., Chen, Y., Peng, X.,
2012. Subcritical co-solvents extraction of lipid from wet microalgae pastes of
Nannochloropsis sp. Eur. J. Lipid Sci. Technol. 114, 205212.
Cooney, M., Young, G., Nagle, N., 2009. Extraction of bio-oils from microalgae. Sep.
Purif. Rev. 38, 291325.
DOca, M.G.M., Vigas, C.V., Lemes, J.S., Miyasaki, E.K., Morn-Villarreyes, J.A.,
Primel, E.G., Abreu, P.C., 2011. Production of FAMEs from several microalgal
lipidic extracts and direct transesterification of the Chlorella pyrenoidosa.
Biomass Bioenergy 35, 15331538.
Daroch, M., Geng, S., Wang, G., 2013. Recent advances in liquid biofuel production
from algal feedstocks. Appl. Energy 102, 13711381.
Dejoye Tanzi, C., Abert Vian, M., Chemat, F., 2013. New procedure for extraction of
algal lipids from wet biomass: a green clean and scalable process. Bioresour.
Technol. 134, 271275.
Doan, T.-T.Y., Obbard, J.P., 2011. Improved Nile Red staining of Nannochloropsis sp.
J. Appl. Phycol. 23, 895901.
Dong, T., Knoshaug, E.P., Pienkos, P.T., Laurens, L.M.L., 2016. Lipid recovery from wet
oleaginous microbial biomass for biofuel production: a critical review. Appl.
Energy 177, 879895.
Fang, X., Fei, X., Sun, H., Jin, Y., 2016. Aqueous enzymatic extraction and
demulsification of camellia seed oil (Camellia oleifera Abel.) and the oils
physicochemical properties. Eur. J. Lipid Sci. Technol. 118, 244251.
Frhlich, H., Gasser, K., Gaul, S., Grtzner, T., Strube, J., 2015. Development of
purification concepts for nutraceuticals from algae. Part I: characterization of
Emulsifying Components. Chem. Eng. Technol. 38, 20252034.
Ghasemi Naghdi, F., Gonzlez Gonzlez, L.M., Chan, W., Schenk, P.M., 2016. Progress
on lipid extraction from wet algal biomass for biodiesel production. Microb.
Biotechnol. 9, 718726.
Grima, E.M., Gonzlez, M.J.I., Gimnez, A.G., 2013. Solvent extraction for microalgae
lipids. In: Borowitzka, M.A., Moheimani, N.R. (Eds.), Algae for Biofuels and
Energy. Springer Netherlands, Dordrecht, pp. 187205.
Hannon, M., Gimpel, J., Tran, M., Rasala, B., Mayfield, S., 2010. Biofuels from algae:
challenges and potential. Biofuels 1, 763784.
Huang, G., Chen, F., Wei, D., Zhang, X., Chen, G., 2010. Biodiesel production by
microalgal biotechnology. Appl. Energy 87, 3846.
Huang, W.-C., Kim, J.-D., 2013. Cationic surfactant-based method for simultaneous
harvesting and cell disruption of a microalgal biomass. Bioresour. Technol. 149,
579581.
799
Huang, Y., Zhang, D., Xue, S., Wang, M., Cong, W., 2016. The potential of microalgae
lipids for edible oil production. Appl. Biochem. Biotechnol. 180, 438451.
Hussain, J., Liu, Y., Lopes, W.A., Druzian, J.I., Souza, C.O., Carvalho, G.C., Nascimento,
I.A., Liao, W., 2015. Effects of different biomass drying and lipid extraction
methods on algal lipid yield, fatty acid profile, and biodiesel quality. Appl.
Biochem. Biotechnol. 175, 30483057.
Lai, Y.S., Francesco, F.D., Aguinaga, A., Parameswaran, P., Rittmann, B.E., 2016.
Improving lipid recovery from Scenedesmus wet biomass by surfactant-assisted
disruption. Green Chem. 18, 13191326.
Lai, Y.S., Zhou, Y., Martarella, R., Wang, Z., Rittmann, B.E., 2017. Synergistic
integration of C12C16 cationic surfactants for flocculation and lipid
extraction from chlorella biomass. ACS Sustain. Chem. Eng. 5, 752757.
Lesieur, S., Grabielle-Madelmont, C., Mnager, C., Cabuil, V., Dadhi, D., Pierrot, P.,
Edwards, K., 2003. Evidence of surfactant-induced formation of transient pores
in lipid bilayers by using magnetic-fluid-loaded liposomes. J. Am. Chem. Soc.
125, 52665267.
Li, Q., Liu, D., Wu, P., Song, L., Wu, C., Liu, J., Shang, X., Yan, Z., Subhan, F., 2016. In-
depth insight into the chemical composition of bio-oil from hydroliquefaction
of lignocellulosic biomass in supercritical ethanol with a dispersed Ni-based
catalyst. Energy Fuels 30, 52695276.
Lichtenberg, D., Ahyayauch, H., Alonso, A., Goi, F.M., 2013. Detergent solubilization
of lipid bilayers: a balance of driving forces. Trends Biochem. Sci. 38, 8593.
Mercer, P., Armenta, R.E., 2011. Developments in oil extraction from microalgae.
Eur. J. Lipid Sci. Technol. 113, 539547.
Opatowski, E., Kozlov, M.M., Pinchuk, I., Lichtenberg, D., 2002. Heat evolution of
micelle formation, dependence of enthalpy, and heat capacity on the surfactant
chain length and head group. J. Colloid. Interface. Sci. 246, 380386.
Pragya, N., Pandey, K.K., Sahoo, P.K., 2013. A review on harvesting, oil extraction and
biofuels production technologies from microalgae. Renewable Sustain. Energy
Rev. 24, 159171.
Samori, C., Lopez Barreiro, D., Vet, R., Pezzolesi, L., Brilman, D.W.F., Galletti, P.,
Tagliavini, E., 2013. Effective lipid extraction from algae cultures using
switchable solvents. Green Chem. 15, 353356.
Schuchardt, U., Sercheli, R., Vargas, R.M., 1998. Transesterification of vegetable oils:
a review. J. Braz. Chem. Soc. 9, 199210.
Schwenzfeier, A., Helbig, A., Wierenga, P.A., Gruppen, H., 2013. Emulsion properties
of algae soluble protein isolate from Tetraselmis sp. Food Hydrocoll. 30, 258
263.
Seo, Y.H., Sung, M., Oh, Y.-K., Han, J.-I., 2015. Lipid extraction and esterification for
microalgae-based biodiesel production using pyrite (FeS2). Bioresour. Technol.
191, 420425.
Sheng, J., Vannela, R., Rittmann, B.E., 2011. Evaluation of methods to extract and
quantify lipids from Synechocystis PCC 6803. Bioresour. Technol. 102, 1697
1703.
Shinoda, K., Kunieda, H., 1973. Conditions to produce so-called microemulsions:
Factors to increase the mutual solubility of oil and water by solubilizer. J.
Colloid Interface Sci. 42, 381387.
Ulloa, G., Coutens, C., Sanchez, M., Sineiro, J., Fabregas, J., Deive, F.J., Rodriguez, A.,
Nunez, M.J., 2012. On the double role of surfactants as microalga cell lysis
agents and antioxidants extractants. Green Chem. 14, 10441051.
Urrutia, C., Sangaletti-Gerhard, N., Cea, M., Suazo, A., Aliberti, A., Navia, R., 2016.
Two step esterificationtransesterification process of wet greasy sewage sludge
for biodiesel production. Bioresour. Technol. 200, 10441049.
Wu, C., Xiao, Y., Lin, W., Li, J., Zhang, S., Zhu, J., Rong, J., 2017. Aqueous enzymatic
process for cell wall degradation and lipid extraction from Nannochloropsis sp.
Bioresour. Technol. 223, 312316.
Xiao, Y., He, P., Cheng, W., Liu, J., Shan, W., Song, H., 2016. Converting solid wastes
into liquid fuel using a novel methanolysis process. Waste Manage. 49, 304
310.
Yan, Y., Xiong, W., Li, X., Lu, T., Huang, J., Li, Z., Fu, H., 2007. Molecular packing
parameter in bolaamphiphile solutions: adjustment of aggregate morphology
by modifying the solution conditions. J. Phys. Chem. B 111, 22252230.