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ARTICLE

Comparison of cell disruption techniques prior to lipid extraction

from Scenedesmus sp. slurries for biodiesel production using

Green Chemistry Accepted Manuscript

Received 00th January 20xx,
Accepted 00th January 20xx
liquid CO2
DOI: 10.1039/x0xx00000x K.J. Vinera,b, P. Champagneb, P.G. Jessopa,*
Published on 23 August 2018. Downloaded on 8/31/2018 6:22:39 AM.

www.rsc.org/ Microalgae have long been considered an ideal potential feedstock for the production of biodiesel because of their fast
growth rates, high productivities, and high intracellular lipid content. Conventional extraction methods generally utilize
high pressures, halogenated organic solvents, dried algae, or long extraction periods, leading to high energy/capital costs
to achieve acceptable microalgal lipid yields. For this reason, liquid CO2 has emerged as an innovative, greener extraction
technique that offers the benefits of utilizing a lower pressure (150 bar) and temperature (25 °C), and lower energy/capital
costs than supercritical CO2. However, obtaining complete access to the lipids contained in microalgal slurries continues to
present a significant challenge. This study investigates mechanical and chemical cell disruption techniques, including
ultrasonication, microwave radiation, grinding with liquid N2, osmotic shock, cooling, and freeze-drying, prior to the
extraction of neutral lipids (NL) and free fatty acids (FFA) from Scenedesmus sp. slurries. The highest NL/FFA yield obtained
was roughly 9.6 wt% when microwave radiation was applied, compared to a total NL/FFA yield of 13.2 wt% obtained by
Soxhlet extraction.

Introduction glycoprotein matrix, providing the cells with a formidable defense

5
against the environment. Efficient extraction of desirable lipids
from dried microalgae is facile because the drying process breaks
Biodiesel, primarily composed of fatty acid methyl esters down the cell walls. However, the enormous energy cost of drying is
(FAMEs), is a potentially greener and more sustainable alternative prohibitive, so the lipids must be extracted from microalgal slurries.
to fossil fuel-derived diesel. The advantages include If the cell walls in such slurries are intact, then it is difficult to
biodegradability, renewability, and lower emissions of CO, SOx, and extract industrially useful quantities of lipids from microalgal
1
particulate matter. Importantly, the combustion of biodiesel slurries difficult, requiring the use of energy intensive extraction
releases similar amounts of CO2 to comparable fossil fuels, but CO2 techniques, leading to higher economic and environmental costs.
is consumed in the growth of the biomass, which reduces the net Therefore, a low energy cell disruption method preceding lipid
CO2 emissions. extraction from wet microalgae is required to minimize those costs.
Microalgae represent an ideal feedstock for the production of Some of the main extraction techniques employed in the
renewable biodiesel because of their rapid growth rates, high recovery of microalgal lipids are organic solvent extraction at
2
productivities, and high intracellular lipid content. Moreover, they moderate temperatures, extraction at elevated temperatures
can be grown on marginal or infertile lands, thereby avoiding (Soxhlet extraction), and extraction with supercritical CO2 (scCO2).
6
competition with the production of agricultural food crops, as well The environmental and economic costs of conventional organic
as grown in wastewater, providing nutrient (nitrogen and solvent extraction can be minimized by choosing a solvent or co-
3
phosphorus) removal, thereby diminishing pollution. Microalgae solvent that is inexpensive, non-toxic and easily recoverable from
are classified as a third-generation biofuel feedstock, whereby first- both the lipid and aqueous fractions. Current methods often use
generation biofuel feedstocks, edible crops, and second-generation flammable and/or chlorinated solvents, such as n-hexanes or a
biofuel feedstocks, non-edible crops, are unsustainable in the long- chloroform/methanol mixture, which increase risks associated with
term. Overall, biodiesel produced from microalgae may be one of 7
fire, acute toxicity, neurotoxicity, and carcinogenicity. Moreover,
the most sustainable alternatives to fossil fuel-derived diesel.4 the use of these solvents is associated with a poor selectivity for the
However, amongst the major barriers delaying techno- extraction of lipids and free fatty acids. Soxhlet extraction typically
economic viability is the need for energy-efficient extraction of the uses a 2:1 v/v mixture of chloroform and methanol, a temperature
desirable lipids from the microalgal cells due to the rigidity of their 7
of 80 °C, and is conducted for 24 h. This technique extracts the
cell walls. These walls are made up of a polysaccharide and highest yield of extract, but is not economically viable because of
the high-energy consumption, the poor selectivity, and the
environmental concerns associated with the use of chlorinated
solvents. Supercritical CO2 extraction, which is usually conducted at
temperatures ranging from 50-80 °C and pressures of 20-30 MPa, is
8
considered to be a promising and greener extraction approach. It

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offers advantages compared to Soxhlet extraction and conventional 1,4-butanediamine (TMBDA), rather than examining a more
21
organic solvent extraction, including higher selectivity for neutral conventional osmotic shock agent such as NaCl. A switchable
lipids (NLs), shorter extraction times, and facile solvent recovery, osmotic shock agent can reversibly change from an organic solvent
9,10
while avoiding the need for halogenated organic solvents. to a salt in the presence of carbonated water. This salt is used to
However, this technique does require high pressures to obtain create a super-concentrated brine solution that draws water out of
yields comparable to conventional liquid organic solvent the microalgal cells. Using a conventional salt, there are substantial
extractions. As such, the economical production of FAMEs from downstream costs associated with separation of the salt from
microalgal lipids is strongly limited by the energy and capital costs water, whereas a switchable salt can be reverted to its organic
associated with the lipid extraction approaches currently available. solvent form in the presence of Ar or air without the need for
Liquid CO2 (lCO2) has emerged as an innovative extraction conventional separation techniques.
technique that offers many of the same advantages as scCO2, but at This study provides a comparison of different cell disruption
a lower pressure (15 MPa) and temperature (25 °C), thereby techniques to determine which is the most effective for the overall

Green Chemistry Accepted Manuscript

reducing the energy costs.11 Since lCO2 has a similar polarity to release of lipids during the subsequent extraction. Moreover, FAME
scCO2, it could exhibit comparable selectivity towards NLs, higher profiles of Scenedesmus sp. lipid extracts, when subjected to each
than for most organic solvents. The ability of lCO2, with or without of the cell disruption techniques, were characterized to quantify the
methanol, to extract lipids from freeze-dried Botryococcus braunii production of biodiesel-desirable chain lengths.
11
was confirmed by Paudel et al. In the present study, the potential
Published on 23 August 2018. Downloaded on 8/31/2018 6:22:39 AM.

for lCO2 with methanol to extract lipids from Scenedesmus sp.

slurries is examined because methanol is fairly green and is needed Materials and Methods
for the subsequent transesterification step.
Obtaining complete access to intracellular lipids using lCO2 Materials
extraction from microalgal slurries continues to present a significant All chemicals were used as received from the suppliers. Carbon
challenge. For this reason, cell disruption prior to extraction is a dioxide (99.99%) was obtained from Praxair. Methanol of HPLC
potential means to enhance microalgal lipid extraction. Mechanical grade (>99.9%) was obtained from Fisher Scientific. An algal slurry
cell disruption methods include bead milling, high-pressure (20 wt% solids) of the microalgal species Scenedesmus sp. was
homogenization, ultrasonication, autoclave, freeze-drying, and obtained from the National Research Council (NRC), Halifax, Canada
microwave radiation. Non-mechanical methods involve lysing the and was kept frozen at -81 °C. The slurries were thawed (placed
9
microalgal cells with acids, alkalis, enzymes, or osmotic shock. under hot water for 10 min) before being subjected to cell
Ultrasonication, involving the disruption of cell walls by cavitation, disruption methods and/or extraction. “Fresh” algal slurries were
along with a 2:1 v/v mixture of chloroform:methanol was shown to used as received from the NRC within 5 days of receipt, being
12
extract 19 % of total lipids from C. pyrenoidosa. Freeze-drying, stored at 20 °C during that time.
involving the removal of water from frozen microalgae, was used in
a previous study to simultaneously dry and disrupt Botryococcus Cell disruption methods
braunii cells before an extraction using lCO2, which achieved a lipid For each cell disruption technique, 10 g of microalgal slurry
11
yield of 19 % on a dry algal biomass basis. Moreover, freeze- aliquots (20 wt% solids) (or 2 g of dry mass in the case of freeze-
drying facilitates the extraction of soluble proteins and active dried samples) were used and all experiments were performed
13
enzymes from C. vulgaris. Microwave radiation, involving rapid under the conditions specified in Table 1. Experiments were
oscillation of molecules causing friction and heating, using ethanol conducted in duplicate; and a control without cell disruption was
and hexane as extraction solvents was conducted on also performed in duplicate. Prior to cell disruption, the frozen algal
14
Nannochloropsis oculta. It was shown that, under optimal slurry was allowed to thaw for 30 min in a hot water bath.
conditions, the addition of microwave treatment to a biofuel
production process contributed <1 % of energy expenses, while it Ultrasonication
increased lipid extraction 3-fold compared to a control. Osmotic Algal slurry (20 wt%) aliquots were mixed with 20 mL of
shock, involving rapid changes in solute concentration, along with methanol in a 100 mL beaker and sonicated using a sonicator probe
both polar and non-polar organic solvents was noted to increase (Fischer Scientific Sonic Dismembrator Model 500) at amplitudes of
lipid recovery approximately 2-fold when performed on 30 % or 60 % for 15 min.
15
Chlamydomonas reinhardtii slurries. It was shown that cooling
microalgal suspensions below their growth temperatures affected Microwave radiation
the composition of fatty acids in the cell membrane and led to cell Algal slurry (20 wt%) aliquots were mixed with 10 mL of
disruption.16-18 In addition, other methods of cell disruption have methanol or distilled water in a 50 mL round bottom flask and
also been examined, such as hydrothermal disruption on subjected to microwave radiation (CEM Corporation Discover
Haematococcus pluvialis slurries and applying the Fenton reaction LabMate) at 300 W for 15 s followed by 15 min of cooling under
19-20
for disrupting C. vulgaris slurries. ambient laboratory conditions (20 °C, 0.1 MPa). This procedure was
As mentioned previously, work conducted by Paudel et al. repeated 3 times.
examined the ability of lCO2 for the extraction of NLs from freeze-
dried microalgae.11 In this study, mechanical and chemical cell Grinding following freezing with liquid N2
disruption techniques, such as ultrasonication, microwave Algal slurry (20 wt%) aliquots were placed in a pestle and
radiation, grinding with liquid nitrogen, osmotic shock, switchable subjected to liquid N2 until frozen. A mortar was then used to grind
osmotic shock, cooling, and freeze-drying, were investigated prior the frozen biomass for 2 min.
to the extraction of lipids from Scenedesmus sp. slurries using lCO2.
The switchable osmotic shock method was unconventional and Osmotic shock
assessed a switchable osmotic shock agent, N,N,N’,N’-tetramethyl-

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Algal slurry (20 wt%) aliquots were subjected to 20 mL of 60 % was allowed to seep through. An aliquot (25 mg) of extract was
w/v NaCl in distilled water in a 100 mL beaker. This beaker was diluted in 1 mL of hexanes and placed on the preconditioned
placed on a stir plate overnight. This high concentration of solute cartridge without vacuum. The cartridge was then eluted with three
outside the algal cells has been reported to cause the rupture of the different solvent systems and 3 individual sample fractions were
15
cell walls. collected. Fraction A collected NLs using 5 mL of 17:3 (v/v)
hexanes:ethyl acetate. Fraction B collected the chlorophyll,
pigments, and other extracted constituents considered not to be
Switchable osmotic shock useful in the production of FAMEs, using 4 mL of 23:1 (v/v)
Algal slurry (20 wt%) aliquots were subjected to 20 mL of 60 % chloroform:methanol. Fraction C collected free fatty acids (FFAs)
w/v TMBDA in distilled water in a 100 mL beaker. Then, CO2 was using 3 mL of 46:1 (v/v) ethyl ether:acetic acid. The fractions were
bubbled through this mixture at ambient laboratory conditions (20 placed on a rotary evaporator to remove residual solvent and
°C, 0.1 MPa) using a gas dispersion tube for 2 h, and placed on a stir allowed to air-dry for 3 h. The weight of each of the three fractions

Green Chemistry Accepted Manuscript

plate overnight. TMBDA is a switchable salt, which means that in was measured gravimetrically, using a modified procedure from
22
the presence of carbonated water, it transforms from an uncharged Bodennec et al. Moreover, Fraction D consisted of the remaining
solute to a bicarbonate salt. extract in the cartridge and was mathematically added to Fraction
B. SPEs were similarly performed on the extracts obtained from
Cooling Soxhlet extraction.
Published on 23 August 2018. Downloaded on 8/31/2018 6:22:39 AM.

Algal slurry (20 wt%) aliquots were placed in 20 mL plastic vials

and stored in a refrigerator (4 °C) to cool in the dark overnight. Soxhlet extraction
11
Based on a modified procedure reported by Paudel et al.,
Freeze-drying duplicate Soxhlet extractions were performed on the previously
Algal slurry (20 wt%) aliquots were placed in 20 mL plastic vials, frozen microalgal slurries (20 wt% solids) and were employed as a
frozen in liquid N2, and freeze-dried overnight (12-15 h) using a reference to assess the efficiency of the lCO2 extraction with and
ThermoSavant ModulyoD freeze-dryer. The freeze-dried powder without cell disruption. Moreover, duplicate Soxhlet extractions
was stored under ambient laboratory conditions (20 °C, 0.1 MPa). were performed on the fresh microalgal slurries (20 wt% solids) to
determine whether freezing changed either the yield of lipid extract
Extraction using lCO2 or the resulting FAME profiles. In this procedure, 250 mg of oven-
A lCO2 tank was connected to a JASCO PU-980 Intelligent HPLC dried algal biomass (95 wt%) was placed in a glass thimble and
pump (Figure 1). The pump was then connected to a 160 mL extracted using 150 mL of a 2:1 v/v mixture of chloroform:methanol
continuously-stirred tank reactor (CSTR; Parr T316SS stainless steel in a Soxhlet extractor placed in an oil bath at 80 °C. After a 24 h
vessel), which contained the cell-disrupted algal slurry, a magnetic extraction period, the lipid extracts were placed on a rotary
stir bar, and 20 mL of methanol. A back-pressure regulator (BPR) evaporator to remove residual solvent and allowed to air-dry for 3
was attached to the CSTR to maintain a constant pressure of 15 h. The weights of the total lipid extracts were measured
MPa throughout the extraction. In a typical extraction using lCO2, gravimetrically and reported on a dry mass basis.
the biomass was contacted with methanol in the CSTR for 3 h and
then lCO2 was pumped from the tank, via the JASCO pump, into the FAME preparation and analysis
vessel, and from thence through the BPR and into a 125 mL Acid-catalysed methanolysis was carried out to prepare FAME
Erlenmeyer flask (sample collection) containing 10 mL of acetone. from the microalgal extracts. The methanolysis procedure was
23
The sample collection flask was connected to the BPR via a modified from Lam and Lee. Briefly, 50 mg of microalgal extract
stainless-steel tube (1 mm diameter, 5 cm long) that extended into was placed in a 50 mL round bottom flask along with 11 mg of
the acetone solution. The acetone was then removed from the concentrated sulfuric acid, 1.5 mL of methanol, 1 mL of
extract using a rotary evaporator. All extractions were conducted in tetrahydrofuran, and a magnetic stir bar. The round bottom flask
duplicate at room temperature (20 °C) for approximately 3 h. was then connected to a condenser. The system was placed on a
hot plate at 90 °C for 3 h under continuous stirring. After 3 h, the
mixture was neutralized with sodium bicarbonate and FAMEs were
extracted with hexanes using a separatory funnel. The extracted
FAMEs were analyzed using a Perkin Elmer Clarus 680 gas
chromatograph equipped with a flame ionization detector and
Thermo Scientific TG-Polar column. The oven temperature was
initiated at 50 °C, for 5 min, raised to 260 °C at a rate of 7 °C/min
and held at 260 °C for 5 min. The injector and detector
temperatures were 260 °C. Helium was used as the carrier gas. The
FAME peaks in the samples were identified by comparing their
TM
retention times with those of a standard mixture (Supelco 37
Figure 1. Process flow diagram for the extraction of microalgal lipids component FAME mix, Sigma-Aldrich).
using lCO2.
Fluorescence microscopy using Nile Red
To effectively monitor cell disruption, Nile Red was used to
Solid Phase Extraction (SPE) 24
fluorometrically determine the NL content in microalgal cells. Nile
Microalgal extract obtained from the extractions using lCO2 was Red was diluted in acetone (10 μg/mL) and 100 μL was added to
fractionated using SupelcleanTM LC-NH2 (500 mg) cartridges. A samples of Scenedesmus sp., placed on microscope slides with their
cartridge was placed on a vacuum manifold and 6 mL of hexanes respective cover slips, before and after cell disruption. These

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samples were then observed under a Leica fluorescence microscope

(40x magnification) containing a rhodamine filter with an excitation The lowest NL+FFA yield of 2.8±0.1 % was obtained using
and emission of 520 nm and 650 nm, respectively. Photographs grinding following freezing with liquid N2 as the cell disruption
were taken using AxioVision 4.7 software. method. Freeze-drying, ultrasonication (30 and 60 % amplitude),
cooling, microwave in the presence of methanol, osmotic shock,
and switchable osmotic shock achieved NL+FFA yields of
Results and Discussion approximately 4.3-5.1 %, similar to the yields obtained without
prior cell disruption (5.1±0.7 %).
Cell disruption followed by extraction using lCO2
Each of the experiments was performed using the same lipid FAME analysis
extraction method with lCO2 and 20 mL of methanol. These The FAME profiles obtained for the Scenedesmus sp. lipid

Green Chemistry Accepted Manuscript

experiments only differed in the type of mechanical/chemical cell extracts with and without prior cell disruption, and with Soxhlet
disruption method applied prior to lipid extraction to effectively 26
extraction alone are shown in Figure 2. According to Knothe, the
liberate valuable NLs+FFAs from the Scenedesmus sp. slurry. Of all most desirable FAMEs for high quality biodiesel production include
the cell disruption techniques tested (Table 1), microwave radiation the C16 and C18 carbon chain lengths as they typically produce
in the presence of distilled water followed by lipid extraction was 2
cetane numbers (47 minimum), viscosities (1.9-6.0 mm /s at 40 °C),
Published on 23 August 2018. Downloaded on 8/31/2018 6:22:39 AM.

found to be most effective as it extracted NL+FFA fractions of and oxidative stabilities (3 minimum) within the ideal range as
9.6±1.5 % compared to a total available NL+FFA yield of 13.2±0.3 %, stated by ASTM D6751 (standard specifications for biodiesel). In this
on a dry mass basis determined by Soxhlet extraction. In other study, for all the FAME profiles obtained, the sum of C16:0, C16:1,
words, this method extracted 73 % of the total extractable C18:0, C18:1, C18:2, and C18:3 accounted for 75-83 % of the total
NLs+FFAs. However, it should be noted that the Soxhlet extraction composition, with the exception of those extracted after
method also extracted far more of the other constituents such as ultrasonication (69 %). The relative FAME proportions varied
phospholipids. The success with microwaves was consistent with a depending on the disruption technique used. For instance, both
study by Lee et al. which reported that microwave radiation FAME profiles for cooling and no cell disruption contained ~82 %
followed by a lipid extraction involving a 1:1 v/v mixture of C16 and C18 carbon chain lengths, but cooling exhibited higher
chloroform and methanol produced the highest lipid extract yield amounts of C16:0, C18:0, C18:2, and C18:3, while no cell disruption
21
(11 %) from Scenedesmus sp. In our experiments, microwave displayed higher amounts of C16:1 and C18:1. FAME profiles for
radiation with 10 mL of water added to the microalgal slurry gave a extraction using lCO2 and 20 mL of methanol, without prior cell
NL+FFA yield that was roughly double (9.6±1.5 %) that obtained disruption (Figure 2b) and microwave radiation with the addition of
after microwave radiation with the addition of 10 mL of methanol 10 mL of water (Figure 2c) were quite similar. The FAME profiles
to the microalgal slurry (4.4±0.7 %). This was likely because water is confirmed that Scenedesmus sp. is a desirable feedstock containing
a more polar solvent than methanol and microwave radiation heats a high fraction of FAMEs that would be beneficial in biodiesel
25
polar solvents more effectively. production.
It is also important to note that the FFAs extracted through the
Soxhlet extraction technique is always lower than the
corresponding ones using lCO2 and prior cell disruption methods.
This is because there is very little water and no CO2 during the
Soxhlet extraction technique, but in all other cases the acidity
caused by the combination of water and high-pressure CO2 can
promote acid-catalyzed hydrolysis of the esters.

a
Table 1. Extraction yields obtained after various cell disruption methods.

Total NL and Yield of algae lipid components

Cell disruption Cell disruption solvent b FFA yields (wt% of dry algae)
Extraction method
method (20 mL) (wt% of dry
NL FFA Other
algae)
c
Oven-drying None Soxhlet 13.2 ± 0.3 12.3 ± 0.3 0.9 ± 0.0 9.3 ± 0.3
None None lCO2/MeOH 5.1 ± 0.7 1.5 ± 0.4 3.6 ± 0.6 1.0 ± 0.3
Freeze-drying None lCO2/MeOH 4.7 ± 0.4 1.4 ± 0.4 3.3 ± 0.1 1.6 ± 0.4
Ultrasonication (30%) Methanol lCO2/MeOH 5.1 ± 0.1 1.0 ± 0.0 4.1 ± 0.1 0.6 ± 0.1
Ultrasonication (60%) Methanol lCO2/MeOH 4.3 ± 0.2 0.8 ± 0.1 3.5 ± 0.2 0.3 ± 0.1
Cooling None lCO2/MeOH 4.5 ± 0.4 1.2 ± 0.4 3.3 ± 0.2 1.3 ± 0.7
Microwave Water lCO2/MeOH 9.6 ± 1.5 5.5 ± 1.2 4.1 ± 0.9 2.5 ± 0.7
Microwave Methanol lCO2/MeOH 4.4 ± 0.7 1.5 ± 0.3 2.9 ± 0.6 1.2 ± 0.3
Liq N2 grinding None lCO2/MeOH 2.8 ± 0.1 0.5 ± 0.1 2.3 ± 0.1 0.3 ± 0.1
Osmotic shock 60 % w/v NaCl lCO2/MeOH 3.2 ± 0.8 1.2 ± 0.2 2.0 ± 0.8 1.1 ± 0.4
Switchable osm. shock 60 % w/v TMBDA lCO2/MeOH 3.2 ± 0.3 1.4 ± 0.3 1.8 ± 0.1 2.6 ± 0.4
a b
Each value represents the mean ± S.D. (n=2). All extractions were performed with 10 g of Scenedesmus sp. slurry (20 wt% solids). After
cell disruption, the extraction process was run for 3 h at 25 °C using lCO2 (15 MPa) and 20 mL methanol except where noted. c The Soxhlet
extraction was performed using 150 mL of a 2:1 v/v mixture of chloroform:methanol in a Soxhlet extractor at 80 °C for 24 h.

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a)

40%

% total FAME composition

35%
30%
25%
20%
15%
10%
5%

Green Chemistry Accepted Manuscript

0%
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b)

40%
% total FAME composition

35%
30%
25%
20%
15%
10%
5%
0%
C10:0
C11:0
C12:0
C13:0
C14:0
C14:1
C15:0
C15:1
C16:0
C16:1
C17:0
C17:1
C18:0
C18:1
C18:2
C18:3
C20:0
C20:2
C20:3
C20:4
C20:5
C21:0
C22:0
C22:1

C22:6
C23:0
C24:0
C24:1
C22:2
c)

40%
% total FAME composition

35%
30%
25%
20%
15%
10%
5%
0%
C10:0
C11:0
C12:0
C13:0
C14:0
C14:1
C15:0
C15:1
C16:0
C16:1
C17:0
C17:1
C18:0
C18:1
C18:2
C18:3
C20:0
C20:2
C20:3
C20:4
C20:5
C21:0
C22:0
C22:1

C22:6
C24:0
C24:1
C22:2

Figure 2. FAME profiles of Scenedesmus sp. upon a) Soxhlet extraction with a 2:1 v/v mixture of chloroform:methanol,
b) extraction using lCO2 and 20 mL of methanol, without prior cell disruption, and c) microwave radiation with the
addition of 10 mL of water to the microalgal slurry followed by extraction using lCO2 and 20 mL of methanol.

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Fresh vs. frozen microalgae Both the extract yields and percentage of NLs extracted did not
It is common in experimental studies of algae to perform all appear to be substantially different. The fresh and frozen
experiments upon a single batch of algae to avoid batch-to-batch microalgal slurry extracts contained 1.0±0.3 % 1.5±0.4 % of NLs,
variation in lipid content and other properties. However, a single on a dry mass basis, respectively. Therefore, freezing the algae
batch of algae cannot be stored at room temperature over the during storage did not significantly affect the extraction results.
many weeks required for such a study without the algae undergoing
changes. Therefore, the algae batch must be stored in a frozen
state. However, the freezing process could cause some cell
disruption and thereby alter the results of the study. We therefore 7
sought to determine whether extraction tests on algae that had 6
been frozen would have different results from tests on fresh algae

Extract yield (%)

from the same batch. 5

Green Chemistry Accepted Manuscript

To determine whether freezing the microalgae would 4 Other
significantly affect the extract yields and FAME profiles, as was
3 FFA
observed through cell disruption, Soxhlet extractions and SPEs were
conducted on both fresh and frozen Scenedesmus sp. (20 wt%). The NL
2
results are shown in Figure 3. In order to minimize cell disruption
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during the freezing process, flash-freezing was performed. 1

0
25 Fresh Frozen
Figure 4. Fraction of total extract yield (dry mass) achieved for
Total extract yield (%)

20
Scenedesmus sp. (fresh and frozen). Liquid CO2 extractions were
conducted at 20 °C for 3 h in the presence of 20 mL of methanol
15 Other and performed in duplicate (n=2). SPE was used to determine the
FFA percentage of NLs, FFAs, and other constituents (Other)
10 recovered in the lCO2 and methanol extract. Error bars are from
NL SPE performed in duplicate (n=2).
5
Fluorescence microscopy using Nile Red
0 Fluorescence microscopy was used to visually assess whether
Fresh Frozen cell disruption had taken place. Nile Red dye, when in the presence
of a lipid-rich environment, can be intensely fluorescent. When it
Figure 3. Fraction of total extract yield (dry mass) achieved for
encounters polar membrane lipids, it fluoresces a deep red and
Scenedesmus sp. (fresh and frozen). Soxhlet extractions were 27
upon contact with NLs, it exhibits a strong yellow-gold emission.
conducted at 80 °C for 24 h using a 2:1 (v/v) chloroform:methanol
Therefore, yellow fluorescence should only be visible when
and performed in duplicate (n=2). SPE determined the
microalgal cell walls were disrupted prior to the dye application.
percentage of NLs, FFAs, and other constituents (Other)
As can been seen from the images taken prior to all cell
recovered in the Soxhlet extract. Error bars are from SPE
disruption applications (left images in Fig. 5), red autofluorescence
performed in duplicate (n=2).
due to the presence of chlorophyll, deep red fluorescence upon
contact with the polar membrane lipids, and no yellow fluorescence
The total extract yields obtained for fresh and frozen were observed in each of the samples. Thus, the results would
Scenedesmus sp., respectively, were 21±1.0 % and 22.5±0.5 %, on a suggest that storage at -81 °C did not cause substantial cell
dry mass basis. Therefore, freezing Scenedesmus sp. did not disruption. After microwave radiation (Fig. 5d) and grinding
significantly affect the total extract yield. The NL+FFA yield was following freezing with liquid N2 (Fig. 5e), yellow fluorescence was
slightly higher for the sample that had been frozen (13.2±0.6 % vs. detected. Microwave radiation with the addition of 10 mL of water
11.1±1.1 % for fresh), but the difference was similar in magnitude to the microalgal slurry appeared to cause the greatest amount of
to the experimental error. The only significant difference was a cell disruption, which was consistent with the higher extract yields
greater yield of NL from the frozen rather than fresh algae. The measured (Table 1). Grinding following freezing with liquid N2
extract from the fresh microalgal slurry contained 8.2±0.2 % of NLs, appeared to cause the second greatest amount of cell disruption;
on a dry mass basis, while the extract from algae that had been however, after lipid extraction, it displayed the lowest yields of NLs
frozen contained 12.3±0.1 %. This demonstrated that freezing and FFAs (Table 1). These lowered yields could be due to the
Scenedesmus sp. could positively affect the total amount of NLs additional distilled water used for transferring the microalgal slurry
extracted. from one vessel to another, causing increased hinderance in the
mass transfer of the NLs and FFAs during the extraction process.
After freeze-drying, cooling, and switchable osmotic shock (Figures
To further evaluate the effect of freezing the microalgae on 5a, b, f), yellow fluorescence was not observed, which would
lipid extraction and composition, lCO2 extractions and SPEs were suggest that the cell walls were not disrupted enough for the Nile
conducted on both fresh and previously frozen (-81 °C) Red dye to interact with the intracellular NLs.
Scenedesmus sp. (20 wt% slurries). The extract yields obtained Microwave treatment is potentially more economical compared
from the fresh and frozen Scenedesmus sp. slurries, respectively, to conventional thermal treatments because a shorter treatment
were 5.3±0.6 % and 6.0±1.0 %, on a dry mass basis (Figure 4). time is required, it is more energy efficient, and it has lower-

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14,28
operating costs. For these reasons, this technique could possibly
be used industrially; however, some disadvantages include the
capital costs associated with the equipment and difficulties in
28
controlling the temperature. Ali and Watson demonstrated that
using 5 minutes of 1021 W microwave radiation on microalgal
slurries (1 g of dry algae with 5 mL of distilled water) would
contribute <1 % of the total energy process costs, while increasing
14
lipid extraction 3-fold in comparison to a control. They also found
that the energy consumption to dry the microalgal slurry (23 wt%) e)
accounted for roughly 80 % of the total energy required to make
14
biodiesel. In the case of the results presented in the current study,
microwave radiation was applied at 3 intervals of 15 seconds each

Green Chemistry Accepted Manuscript

using 300 W of power, which would therefore contribute less than 1
% to the total energy expenses and could lead to a 2-fold increase in
lipid extract yield compared to the control. In the study by Lee et
20µm
al., a variety of cell disruption methods were assessed, including 20µm
microwave radiation, bead beating, autoclave, sonication, and
Published on 23 August 2018. Downloaded on 8/31/2018 6:22:39 AM.

osmotic shock followed by a lipid extraction (1:1 v/v mixture of

chloroform and methanol) on three wet microalgal slurries of f)
21
Scenedesmus sp., Chlorella vulgaris, and Botryococcus sp. Among
the cell disruption methods investigated, they reported that
microwave radiation prior to solvent lipid extraction achieved the
highest lipid yield and appeared to be the most efficient for all
three microalgal species.
20µm
20µm

ba)
Figure 5. Fluorescence microscopy images of Scenedesmus sp.
20µm before (left) and after (right) cell disruption: a) freeze-drying, b)
ultrasonication (30%), c) cooling, d) microwave in the presence of
water, e) grinding following freezing with liquid N2, and f)
switchable osmotic shock using Nile Red. Red autofluorescence
20µm indicates presence of chlorophyll and yellow fluorescence indicates
NL.

b)
Conclusions
Microwave radiation in the presence of distilled water
exhibited the highest potential for releasing NLs and FFAs from
Scenedesmus sp. prior to extraction using lCO2 and methanol.
All other cell disruption techniques produced extract yields no
greater than that achievable without prior cell disruption.
20µm 20µm Freezing alone was as ineffective as most of the cell disruption
2 2 techniques investigated. Fluorescence microscopy showed
qualitatively that microwave treatment caused the greatest
c) 20µm degree of cell disruption. This was then selected as the
20µm 20
2 pretreatment method of choice that was allowed the highest
extract yields. FAME composition profiles showed that C16 and
C18 made up 69-83 % of the total FAMEs identified, regardless
of the method of cell disruption, indicating that Scenedesmus
sp. could be employed as a valuable feedstock in the
production of biodiesel.
Further experimentation and a life cycle assessment are
required to determine the energy balance for the extraction
with microwave pretreatment and whether similar or better
efficiency could be obtained with conventional heating.
d) Microwave heating is generally more energy efficient than
conventional heating for many applications,29-31 especially at
31
larger scales.

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Overall, these findings regarding the use of cell disruption Morón-Villarreyes, J. A.; Primel, E. G.; Abreu, P. C.
to extract NL/FFA from microalgal slurries can be applied to Production of FAMEs from Several Microalgal Lipidic
the extraction of other compounds and from other organisms, Extracts and Direct Transesterification of the Chlorella
such as glucose from macroalgae for bio-jet fuel production Pyrenoidosa. Biomass and Bioenergy 2011, 35 (4), 1533–
and fatty acids from E. Coli for biodiesel production.
1538.
(13) Unterlander, N.; Champagne, P.; Plaxton, W. C.
Lyophilization pretreatment facilitates extraction of soluble
Acknowledgements
proteins and active enzymes from the oil-accumulating
We thank the Ontario Ministry of Research Innovation – microalga Chlorella vulgaris. Algal Research 2017, 25, 439-
Ontario Research Fund, Natural Sciences and Engineering 444.
Research Council (NSERC), BioFuelNet, and the Canada
(14) Ali, M.; Watson, I. A. Microwave Treatment of Wet Algal
Research Chairs program for their funding support. We also

Green Chemistry Accepted Manuscript

thank the National Research Council (NRC) for providing Paste for Enhanced Solvent Extraction of Lipids for
microalgae. Biodiesel Production. Renew. Energy 2015, 76, 470–477.
(15) Yoo, G.; Park, W. K.; Kim, C. W.; Choi, Y. E.; Yang, J. W.
Direct Lipid Extraction from Wet Chlamydomonas
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