Enhanced Lipid Extraction From Microalgae in Biodiesel Production
Enhanced Lipid Extraction From Microalgae in Biodiesel Production
Enhanced Lipid Extraction From Microalgae in Biodiesel Production
Abstract
In order to secure more effective lipid extraction method, this research investigated new SCIENTIFIC PAPER
lipid extraction method using laser with absorbent and sought its optimum operation
control. In addition, this study compared lipid extraction efficiency and FAME conversion UDC 662.756.3:66.061.3:54
rate between laser extraction method at optimum condition and existing extraction
method. Results from experiments for optimizing lipid extraction method using laser
showed that the maximum extraction efficiency (81.8%) was attained when using laser Hem. Ind. 71 (2) 167–174 (2017)
with an output capacity of 75Wh/L. Extraction efficiency increased up to 90.8% when mic-
rowave treatment as pretreatment process was conducted. Addition of absorbents during
lipid extraction process with laser showed higher extraction efficiency than laser and
chemical method. It was also found that laser extraction method with absorbent had
higher total fatty acid content (853.7 mg/g oil) in extracted lipid than chemical extraction
method (825.4 mg/g oil). Furthermore, it had the highest FAME conversion rate (94.2%).
Keywords: biodiesel, microalgae, laser, lipid extraction.
Available online at the Journal website: http://www.ache.org.rs/HI/
In the field of new renewable energy, development [3]. In addition, the process can be performed under
of alternative energy using biological resources has very mild conditions at room temperature and atmo-
received great attention, especially biofuel production spheric pressure, and produced biomass can also be
that includes bioethanol and biodiesel as alternative utilized as a useful substance. The production process
transport fuels. Although biodiesel produced through of biodiesel using microalgae is composed of cultivation
trans-esterification reaction has similar physical of microalgae, harvesting biomass, lipid extraction
properties with petroleum-based diesel, it discharges process from algae and trans-esterification reaction of
less contaminant (e.g., particle matter) into extracted oil [4]. Among the aforementioned product-
atmosphere than conventional fossil fuel and reduces ion processes, lipid extraction process has been reg-
carbon dioxide emissions during cultivating oilseed arded as an obstacle to biodiesel production due to the
crop [1]. In addition to the existing biodiesel production high cost problems and low extraction rate [2]. Several
methods from animal and plant extracts, a newer methods such as expeller/press, solvent oil extraction,
method has been developed that utilizes vegetable oil Soxhlet extraction, hydro-cracking, pyrolysis, lique-
extracts such as soybean and palm oil extracts. Doc- faction and hydrogenation have hitherto been used to
umented evidence by Chisti [2] showed that gradual extract lipid from microalgae [5]. However, these
substitution of current transport fuels with biodiesel methods are not only economically inefficient, but also
from vegetable oil extracts has adverse effects such as: very slow. For example, chemical solvent extraction
destruction of ecosystem caused by farmland expan- method, recognized as practically applicable method
sion (agricultural related activities); inflation of grain because of its relatively high extraction rate, requires
price by agflation; food scarcity and soil contamination pretreatment process such as drying and dewatering
by fertilizer. Therefore, current focus has been redir- process of microalgae [6]. Thus, simple and effective
ected on microalgae, characterized as third generation extraction method for biodiesel from microalgae
raw materials for biodiesel. should be developed and the process can be reason-
Biodiesel production using microalgae is an environ- ably efficient if lipid can be directly extracted from
mental friendly method which can biologically remove microalgae in slurry condition [7].
carbon dioxide by photosynthesis, which is the basic In this study, lipid extraction method using laser as
principle of the material circulation in the natural world an environmental friendly and economical method was
developed in order to overcome limitations of chemical
Correspondence: Y.-J. Kim, Department of Environmental and Energy
method. Existing chemical methods utilize highly vol-
Engineering, Kyungpook National University, Daehak-ro 80, Buk-gu, atile and toxic organic solvent which has low reutil-
College of Engineering Bld. 6 room 509, Daegu, Republic of Korea – ization efficiency [8]. One of the advantages of ext-
41566. racting lipid using laser is that the process can be
E-mail: yjukim@knu.ac.kr
undertaken in slurry status thereby eliminating other
Paper received: 27 March, 2016
Paper accepted: 17 June, 2016
miscellaneous processes. In addition, lipid extracted
https://doi.org/10.2298/HEMIND160327027K from microalgae is composed of triglyceride, chloro-
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M.-G. KIM et al.: LIPID EXTRACTION FROM MICROALGAE IN BIODIESEL PRODUCTION Hem. ind. 71 (2) 167–174 (2017)
phyll and carotene [9]. However, investigation con- 30 min. The weight of the crude lipid obtained from
ducted by Tautorus [9] showed that some components samples was measured as follows:
(chlorophyll and carotene) inhibit transesterification of
triglyceride. Issariyakul and Dalai [10] proposed better 100 ( A − B ) C
Total contents = (1)
method of removing such inhibitors using absorbents. D
The objectives of this study were to obtain opti- where A is the weight of extracted solution after the
mum operation control of lipid extraction method with drying, B is the weight of the weighing bottle, C dilution
laser by analyzing the effect of various factors. Effects rate and D dry weight of sample.
of different pretreatment methods during lipid extract-
ion were analyzed. Effect of laser’s input power cap- Cell disruption
acity was also analyzed. In addition, the effect of add- Cell wall damaging of microalgae, as a pretreatment
ing different absorbents in the slurry during lipid ext- process for facilitating lipid extraction was necessary to
raction and transesterification process were inves- improve extraction efficiency [13]. Thus, ultrasonic-
tigated. Finally, fatty acid methyl ester (FAME) con- ation, microwave and grinding were selected as pre-
version rate based on each method was investigated. treatment for cell disruption and mutually compared in
order to determine optimized pretreatment of lipid
MATERIALS AND METHODS extraction method for this research. Ultrasonication
and microwave treatment were respectively conducted
Cultivation and harvest of microalgae for 10 and 5 min using 28 kHz sonicator (SH-1025,
Scenedesmus sp. which has several advantages such Saehan sonic, Korea) and 2450 MHz microwave oven
as being less contaminated by other microorganisms (MW202LW, LG, Korea).
and having a potential of propagation in the water
Lipid extraction method using laser
containing high concentration of organic materials and
heavy metals, was selected as target microalgae used The laser extraction method used in this study was
for this research [11]. Scenedesmus sp. was isolated designed as shown in Figure 1. The type of laser used
from an indoor raceway pond at Kyungpook National was 445nm blue laser with maximum output power of
University in South Korea. In order to obtain enough 2.5W (Working Voltage: DC 7.4 V, Beam Divergence:
biomass for the production of biodiesel based on <1.2 mRad, Beam Diameter: <1.2 mm @ aperture).
microalgae, each seed culture was used to inoculate Five lasers were installed along the perimeter of the
16L of a commercial liquid fertilizer (1:500 dilution; eradiation tank. In order to maximize the reflectivity of
5.1% N, 10% P2O5 and 5% K2O) in an 18 L transparent laser, mirrors were installed inside the eradiation tank.
polycarbonate bottle and was autotrophically grown at After putting 1.4 L of prepared microalgae slurry into
20 °C with a flow of air bubbles at rate of approx- the eradiation tank, it was radiated by a set of five 2.5 W
imately 2 L/min under cool fluorescent lighting (approx- lasers to produce 25, 50, 75 and 100 W of overall
imately 70 μmol/(m2 s)) with a light: dark cycle of 16:8 energy consumption respectively.
h. After incubating, biomass was harvested by cen-
Analysis of fatty acid profile and extraction efficiency
trifugation at 4615g for 10 min and stored at –70 °C.
In this study, direct trans-esterification was applied
Lipid content analysis of target microalgae to the analysis of the composition and content of fatty
Overall lipid content was extracted from Scene- acid of lipid extracted from microalgae. After 10 mg of
desmus sp. and estimated using modified method of extracted lipid was put into the glass vial, 1 ml of
Bligh and Dyer [12] to calculate the total lipid content. heptadecanoic acid-chloroform solution (Sigma-Aldrich,
Lipids were extracted in the water bath at 65 °C for 1 h, 500 μg/L) as internal standard substance was added.
after 60 ml Chloroform–methanol mixed solvent (2:1 And then 1 ml of methanol and 0.3 ml of sulfuric acid
volume ratio) was added into 1g of sample in the 250 were added respectively. Prepared sample was agit-
ml round bottom flask. After the extraction was com- ated using vortex mixer (KMC-1300V) for 10 min and
plete, it was washed 3 times using 5 ml of chloroform– reacted at 100 °C for 10 min in constant-temperature
–methanol (2:1 volume ratio) with the filtration by water bath. After the sample was cool down at room
using qualitative filter papers (Advantec, No.2) and temperature and blended with 1 ml of distilled water
then evaporated the solvent from extracted mixed sol- with vortexing for 5 min, 1 ml from the bottom of layer
ution in a water bath at 65 °C, and 25 ml of petroleum separated by centrifugation (4000g) was picked out
ether and 15 g of anhydrous sodium sulfate were using syringe. Fatty acid profile analysis of target mic-
added. Petroleum ether layer was separated by centri- roalgae was performed using gas chromatograph
fugation at 3000g for 5 min, after shaking for a minute (Shimadzu Scientific Instruments, Columbia, MD, USA)
and then 10 ml of supernatant was dried at 105 °C for equipped with a FID (Flame Ionization Detector) and HP
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M.-G. KIM et al.: LIPID EXTRACTION FROM MICROALGAE IN BIODIESEL PRODUCTION Hem. ind. 71 (2) 167–174 (2017)
19091B-102 column (25 m×0.2 mm×0.33 µm). The with 5 ml of internal standard and analyzed by GC
composition was identified by comparing the retention (Agilent 6890N). FAME contents of biodiesel derived
times and fragmentation patterns with those for stan- from Scenedesmus sp. was calculated using equation
dards. Mix RM3, Mix RM5, GLC50 and GLC70 (Supelco) below:
were used as the external standard substances. After
analysis of fatty acid content (mg/g oil) of extracted 100 A − AEI CEIVEI
FAME Content = (3)
lipid, fatty acid extraction efficiency was calculated as AEI m
shown below:
In the equation above, ΣA – total area of the peak
a from C14:0 to C24:1, AEI – area of methyl heptadec-
ηF = 100ηL (2)
1000 anoate (ISTD), CEI – concentration (mg/ml) of methyl
heptadecanoate (ISTD), VEI – volume of used methyl
where, ηF is fatty acid extraction efficiency (mass%), ηL heptadecanoate (ISTD), m – the amount of sample
is lipid extraction efficiency and a is fatty acid content analyzed.
in extracted lipid.
Removal of free fatty acid
Biodiesel conversion reaction of extracted lipid
In general, trans-esterification refers to the process
One of the limitations of esterification reaction is of producing FAME by adding the base catalyst to
that it requires large amount of chemical catalyst for triglyceride and reacting with alcohol. However, it has
fatty acid methyl ester (FAME) production [14]. In this been known that free fatty acid present during FAME
experiment, the effect of temperature and input conversion reaction using lipid extracted from micro-
amount of catalyst was investigated. Potassium hydro- algae is the source of saponification and leads to the
xide was selected as a base catalyst for trans-esterific- reduction of reaction yield [15]. Hence, FFA content in
ation and FAME was extracted from prepared fatty acid raw material for biodiesel production should be con-
by using petroleum ether with water in separating fun- verted or removed by esterification since they are
nel. After extraction process, it was washed with dis- impurities in the biodiesel conversion process and have
tilled water more than three times and petroleum an influence on acid value. The removal rate of FFA by
ether was dewatered by anhydrous sodium sulfate. the input amount of sulfuric acid, which was used as
FAME was produced by transpiring and removing suit- acid catalyst in the esterification process, was com-
able amount of remained solution at a constant water pared and analyzed. In general, FFA can be expressed
bath temperature of 65 °C. Content of obtained FAME as % content on the assumption that most oil and fat
was analyzed using European Standard method EN component are composed of oleic acid and it can be
14103. To make an internal standard of 10 mg/ml con- represented as a half of acid value. FFA content of lipid
centration, n-heptane was filled to the marked line of extracted from target microalgae was calculated from
50 ml mess flask added with 500 mg of methyl heptan- acid value obtained by neutralization method. To find
decanoate (C:17:0). Later, 250 ml of sample was mixed acid value, the standard analysis method of EN ISO
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M.-G. KIM et al.: LIPID EXTRACTION FROM MICROALGAE IN BIODIESEL PRODUCTION Hem. ind. 71 (2) 167–174 (2017)
661(animal and vegetable fats and oils – Preparation of polar lipid (e.g., phospholipids and galactolipids). Tri-
test sample) was used. After melting fat by putting acylglycerol is usually used as main raw material for
toluene and isopropyl alcohol at 1:1 ratio, two or three biodiesel production. In order to find out the content of
drops of 1% phenolphthalein solution was added. Later, triacylglycerol which can be converted into biodiesel,
it was measured by titrating with 0.1 M KOH until pink fatty acid composition of Scenedesmus sp. cultivated in
was kept for 30 s. The content of acid value and FFA this research was analyzed by GC.
was calculated using following formula proposed by Results of GC analysis of fatty acid composition
American Oil Chemists Society [16]. showed that Scenedesmus sp. in this study had suitable
56.11Vc composition of fatty acid (C14∼C20) which is necessary
Acidic value = (4) for the biodiesel production and the order of main fatty
m acids was C18:2 > C16:2 > C16:0 (Table 2).
where V is the bulk solution of spent 0.1 M KOH, c
represents molar concentration of KOH (M) and m Table 2. Fatty acid contents and composition of lipids from of
represents the weight of sample. Scenedesmus sp.
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M.-G. KIM et al.: LIPID EXTRACTION FROM MICROALGAE IN BIODIESEL PRODUCTION Hem. ind. 71 (2) 167–174 (2017)
Analysis of pre-treatment process using laser extraction method showed that the maxi-
Ultrasonication and microwave are the most widely mum extraction efficiency (81.8%) was attained when
used methods for cell disruption because of their cap- using laser with an output capacity of 75 Wh/L (Table 4).
acity to generate heat rapidly through amplification of
Table 4. Comparison of extracted lipid amount, extraction
vibration frequency for polar substances such as water yield, and efficiency by consumption energy
for a shorter period of time [18]. However, in the
absence of heating medium, the microalgae will burn if Consumption energy
it is radiated by microwave. For this reason, existing Parameter (Irradiation time, h), W h/L
methods such as chemical and Soxhlet lipid extraction 25 (2) 50 (4) 75 (6) 100 (8)
are cumbersome in nature since they require additional Extracted lipid amount 87 94 109 107
processes such as pouring distilled water onto dried mg/(g cell)
microalgae. This experiment was conducted under opti- Extraction yield, % 8.7 9.4 10.9 10.7
mal operation condition of laser. From experimental Extraction efficiency, % 65.3 70.6 81.8 80.3
results, it was identified that lipid extraction efficiency
by each pre-treatment generally increased at least over Effect of absorbent
10% in accordance with an increasing extraction time
This analysis was conducted with the aim of deter-
(Table 3).
mining the effect of different absorbents at optimal
Table 3. Comparison of lipid extraction efficiency by different operation conditions of laser. Lipid extraction efficiency
pretreatment methods was estimated based on input amount of each absorb-
ent and consumption energy. Three materials were
Time,min selected as target absorbent, namely: activated carbon,
Pre-treatment Parameter
3 5 10 loess and clay. As shown in Table 5, it showed that at
Grinding Total lipid content 80 91 94 relatively low consumption energy (25 W h/L) lipid ext-
mg/(g cell) raction efficiency with loess and clay decreased with an
Extraction efficiency, % 60.1 68.3 70.6 increase of input amount of respective absorbent. This
Ultrasonication Total lipid content 108 118 121 was because the energy from laser was not sufficient
mg/(g cell) enough to penetrate through the absorbent to the cell
Extraction efficiency, % 81.1 88.6 90.8 wall. At 50 W h/L, lipid extraction efficiency when loess
Microwave Total lipid content 91 105 109 and clay were used increased with an increase in input
mg/(g cell) amount of absorbent (up to 0.5 g) and then decreased
Extraction efficiency,% 68.3 78.8 81.8 when the input amount was increased to 1 g. The same
trend was observed at 75 and 100 W h/L. At consump-
Experimental results also showed that lipid extract- tion energy higher than 50 W h/L, lipid decomposition/
ion efficiency obtained using ultrasonication (90.8%) /oxidation could take place.
was higher than that of microwave and grinding for 10 In contrast to previously mentioned absorbents, dif-
min. From these results, we concluded that ultrasonic- ferent trend in lipid extraction efficiency was exhibited
ation was suitable pre-treatment process for lipid ext- by activated carbon. Although correlation between
raction from Scenedesmus sp. using laser. consumption energy and input amount of activated
carbon could not be established, highest lipid extrac-
Optimization of lipid extraction process using laser tion efficiency was obtained with input amount of 0.5 g
Lipid extraction yield and efficiency based on con- and power consumption of 50 W h/L.
sumption energy according to the laser radiation were
Analysis of total fatty acid content
identified using five blue lasers with 2.5 W maximum
output and 445 nm wavelength. In this study, extract- Comparative analysis of Fatty acid content and fatty
ion yield represented total amount of extracted lipid acid extraction efficiency between Soxhlet, laser and
per 1g of microalgae and extraction efficiency indicated laser with absorbent extraction method was analyzed
quantity of extracted lipid per total lipid content of mic- and results are shown in Table 6. The absorbent used in
roalgae in percentage terms. Extraction efficiency inc- this analysis was activated carbon. Fatty acid extraction
reased when the output Power of laser was increased efficiency was calculated by multiplying total fatty acid
up to 75 W. After that, the extraction efficiency dec- content in lipid which was extracted through each
reased when the output power increased from 75 to extraction method by lipid extraction efficiency. Results
100 W. This indicated that there was either decompo- indicated that extraction method using laser with
sition or oxidation of the lipid by laser. Results from absorbent had higher total fatty acid content (853.7
experiments for optimizing lipid extraction method mg/g oil) in extracted fatty acid content than both
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M.-G. KIM et al.: LIPID EXTRACTION FROM MICROALGAE IN BIODIESEL PRODUCTION Hem. ind. 71 (2) 167–174 (2017)
Soxhlet (825.4 mg/g oil) and laser extraction method increased with an increase in the input amount of
(811.4 mg/g oil). catalyst for temperature. At 65 °C, the optimal input
amount of catalyst was 1.5% which accounted for 94.2%.
Table 5. Comparison of lipid extraction efficiency by type of
absorbent used and consumption energy Table 7. FAME content by input amount of catalyst and
reaction temperature
Input amount Consumption energy, W h/L
Absorbent
g 25 50 75 100 Input, mass%
Temperature, °C
Loess 0.3 63.8 90.8 85.6 67.6 0.5 1 1.5
0.5 61.6 92.3 88.6 70.6 45 78.4 85.7 86.9
1 57.8 90.8 87.1 60.8 55 79.7 87.4 89.5
Clay 0.3 65.3 90.2 89.3 70.6 65 83.5 94.1 94.2
0.5 63.8 93.1 92.5 75.8 75 81.7 89.2 90.8
1 59.3 91.6 87.8 64.8
Activated 0.3 66.1 90.8 87.8 61.6 The aforementioned analysis was conducted based
carbon 0.5 67.6 93.8 89.3 60.1 on different extraction methods. Table 6 showed com-
1 63.1 72.1 84.1 88.6 parison of free fatty acid and FAME contents of bio-
diesel using each extraction method. Experimental data
Table 6. Comparative analysis on fatty acid extraction effici- showed that extraction method using laser with abs-
ency and FAME conversion rate of three extraction methods orbent had higher FAME conversion rate (94.2%) than
laser and Soxhlet extraction method. This was due to
Total fatty Fatty acid FAME lower FFA content in extracted lipid when extraction
Extraction Free fatty
acid content extraction conversion
method acid, % process was conducted using laser with absorbent.
mg/(g oil) efficiency, % rate, %
Laser and Soxhlet extraction method had 19.5 and
Laser 811.4 19.5 73.7 93.8 21.4%, respectively.
Laser with 853.7 19.1 80.1 94.2 It is considered that absorbent reduces acidic value
absorbent of lipid since FFA among other components inhibits
Soxhlet 825.4 21.4 65.7 92.7 transesterification by causing saponification with base
catalyst. Therefore, laser extraction method with abs-
Total fatty acid extraction efficiency using laser with orbent could be considered as a suitable process for
absorbent was also higher (80.1%) than both Soxhlet biodiesel production using microalgae.
and laser method. This indicated that 19.9, 26.3 and
34.3% of overall ingredients in extracted lipid couldn’t CONCLUSIONS
be converted to biodiesel by laser with absorbent, laser
and Soxhlet method, respectively. Therefore, laser with In order to overcome problem of existing chemical
absorbent extraction will show higher extraction effi- extraction methods in biodiesel production using mic-
ciency in manufacturing biodiesel. roalgae, more effective lipid extraction method with
laser was developed and its optimum operation control
Comparison of FAME content by each extraction
was sought.
method
Experimental results indicated that lipid content in
FAME content was analyzed based on reaction Scenedesmus sp. was below average because of the
temperature and input amount of catalyst with the aim condition of the culture medium. It was concluded that
of determining optimal condition for transesterific- lipid content tends to increase when microalgae is sub-
ation. Result shown in Table 7 indicated that FAME con- jected to high stress environment culture. Lipid
tent increased by increasing reaction temperature and obtained after extraction contained suitable compo-
catalyst input. In case of reaction temperature, the sition of fatty acid (C14∼C20) which exhibited good
highest FAME content was obtained at 65 °C (83.5%). flow properties at low temperature and poor oxidation
However, at 75 °C the amount of FAME content stability.
obtained decreased. It also revealed that all the pretreatment methods
This was attributed to the fact that this tempera- used for cell disruption showed an increase in lipid ext-
ture (75 °C) was higher than the boiling point of meth- raction over time under optimal operating conditions
anol which will lead to partial evaporation of methanol. of the laser. Ultrasonication was the best pre-treat-
This partial evaporation has negative effect on the ment method for lipid extraction with an overall ext-
catalyst:methanol ratio. Due to this reason, conversion raction efficiency rate of 90.8%.
rate of FAME content was reduced. Results from the With regards to optimization of lipid extraction
amount of input catalyst showed that FAME content using laser, maximum lipid extraction efficiency (81.8%)
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M.-G. KIM et al.: LIPID EXTRACTION FROM MICROALGAE IN BIODIESEL PRODUCTION Hem. ind. 71 (2) 167–174 (2017)
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