Bioresource Technology 181 (2015) 4753

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

Biosynthesis, characterization and enzymatic transesterication of single

cell oil of Mucor circinelloides A sustainable pathway for biofuel
production
Ana K.F. Carvalho, Juan D. Rivaldi, Jayne C. Barbosa, Heizir F. de Castro
Engineering School of Lorena, University of So Paulo, 12602-810 Lorena, So Paulo, Brazil

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

 A Brazilian Mucor circinelloides strain

was found to be a good SCO producer.
 The microwave-assisted EtOH
extraction led to the best extraction
of SCO.
 The SCO showed considerable
amount of monounsaturated fatty
acids.
 The enzymatic ethanolysis of the SCO
achieved high yields of esters.
 Biodiesel properties from SCO are
comparable with those from
vegetable oils.

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

Article history: The lamentous fungus Mucor circinelloides URM 4182 was tested to determine its ability to produce
Received 10 November 2014 single-cell oil suitable for obtaining biodiesel. Cell growth and lipid accumulation were investigated in
Received in revised form 18 December 2014 a medium containing glucose as the main carbon source. A microwave-assisted ethanol extraction
Accepted 19 December 2014
technique (microwave power 6200 W, 5060 C) was established and applied to lipid extraction from
Available online 9 January 2015
the fungal hyphae to obtain high lipid concentration (44%wt) of the dry biomass, which was considerably
higher than the quantity obtained by classical solvent methods. The lipid prole showed a considerable
Keywords:
amount of oleic acid (39.3%wt), palmitic acid (22.2%wt) and c-linoleic acid (10.8%wt). Biodiesel was pro-
Single-cell oil
Mucor circinelloides
duced by transesterication of the single-cell oil with ethanol using a immobilized lipase from Candida
Microwave-assisted extraction antarctica (Novozym 435) as the catalyst. 1H NMR and HPLC analyses conrmed conversion of 93% of
Lipid the single-cell oil from M. circinelloides into ethyl esters (FAEE).
Biodiesel 2015 Elsevier Ltd. All rights reserved.

1. Introduction
The rst generation of biodiesel is currently produced by the
transesterication of triglycerides derived from vegetable oils
and alcohols (methanol or ethanol) using alkaline catalysts. The
limited availability and high cost of the traditional sources of lipid
Corresponding author at: Engineering School of Lorena, University of So Paulo,
Laboratory of Biocatalysis, Estrada Municipal do Campinho, 12602-810 Lorena, So
Paulo, Brazil. Tel.: +55 12 31595149.
E-mail address: heizir@dequi.eel.usp.br (H.F. de Castro).
have motivated searches for new natural sources for the transeste-
rication process. Single-cell oil has received attention as an alter-
native to traditional feedstocks to produce a third-generation
biodiesel (Ho et al., 2014; Leiva-Candia et al., 2014). Lipids can
be accumulated by microorganisms including lamentous fungi,
yeast, bacteria and algae (Khot et al., in press; Santamauro et al.,
2014; Zhang et al., 2014; Da Rs et al., 2013). The production of
biodiesel from algae and cyanobacteria is an important research
topic, and the development of large-scale production processes
has already started in many countries. The production of lipids
from lamentous fungi and yeast as feedstock for biofuels is not

http://dx.doi.org/10.1016/j.biortech.2014.12.110
0960-8524/ 2015 Elsevier Ltd. All rights reserved.
48 A.K.F. Carvalho et al. / Bioresource Technology 181 (2015) 4753
well explored. Single-cell oil is a natural lipid source, and its pro-
duction is not affected by climate changes, location, harvest time
and transport. These factors have an effect on the global cost of
biodiesel produced from vegetable oil (Ratledge, 2004).The compo-
sition of microbial lipids is similar to vegetable oil and animal fats,
and the prole of fatty acids (FAs) includes lauric acid (C12:0),
myristic acid (C14:0), palmitic acid (C16:0), estearic acid (C18:0),
oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3) and
docohexeanoic acid (C22:6) (Khot et al., in press; Vicente et al.,
2009). One of the most promising organisms for lipid production
is the zygomycete fungus Mucor circinelloides. The fungus is rich
in polyunsaturated fatty acids and c-linolenic acid (GLA, 18:3, n-
6), a valuable omega-6 fatty acid that is found mostly in plant-
based oils (Vicente et al., 2010; Xia et al., 2011).
Efcient extraction techniques are required to improve the
competitiveness of microbial lipid production processes. Various
extraction techniques including solvent extraction are used to
isolate biocompounds from natural sources. However, these sol-
vent-based extraction methods are environmentally unsafe, time-
consuming, and difcult to control and can cause undesired
changes in the extractable compounds (Cravotto et al., 2008).
Microwave-assisted ethanol extraction has been used to obtain
essential oil from plants (Cravotto et al., 2008) and represents a
potential method for extraction of lipids from microorganisms
(Loong and Idris, 2014). This method is based on the absorption
of electromagnetic energy by the solvents and cells and its trans-
formation to heat energy. The irradiation provides selective heat-
ing of the sample-solvent mixture with low or imperceptible
changes in lipid composition. In many cases, the microwave heat
may be less damaging than any other extraction methods that
include heating (Carrapiso and Garca, 2000). The advantages of
this method include fast and high quantity lipid recovery and
low solvent volume (Mercer and Armenta, 2011).
The transesterication process for biodiesel production requires
homogeneous and strong base catalysts such as sodium or potas-
sium hydroxide. Heterogeneous catalysts such as enzymes immobi-
lized in inert supports have received attention recently because of
their high protein stability, easy recovery, and ability to be used in
several reaction cycles (Christopher et al., 2014; Zhang et al., 2012).
In this study, the synthesis of single-cell oil from M. circinello-
ides and its extraction using ethanol and microwave radiation were
investigated. A comparison of the extraction yields with other
extraction methods using solvent was also conducted. The lipid
prole and oil properties were analyzed to determine its applica-
bility as feedstock for biodiesel. The single-cell oil was converted
into biodiesel with an enzyme-based heterogeneous catalyst, and
its properties were analyzed according to the standard
specications.
2. Methods
2.1. Chemicals and reagents
Palm (Elaeais guineensis) oil was acquired from AGROPALMA
(Belm, PA, Brazil), and jatropha (Jatropha curcas) oil was obtained
from Instituto Agronmico do ParanIAPAR (Londrina, PR, Brazil).
All the chemicals and reagents were purchased from Sigma
Aldrich and used without any further purication.
2.2. Microorganism and culture conditions
The fungus M. circillenoides f. griseo-cyanus URM-4182 was
tested for its ability to produce lipids suitable as a material for bio-
diesel production. This strain has a documented high lipase activity
(Andrade et al., 2014) and was purchased from the URM culture
collection of Federal University of Pernambuco (Recife, PE, Brazil).
The strain was maintained in potato-dextrose agar at 4 C and
transferred each month. The cultivation was performed in 250-
mL Erlenmeyer asks containing 100 mL of medium with the fol-
lowing composition: glucose, 20 g/L; thiamine, 0.001 g/L; glutamic
acid, 1.5 g/L; nicotinic acid, 0.001 g/L; ammonium sulfate, 1.5 g/L;
and yeast extract, 0.5 g/L. The initial pH of the medium was
adjusted to 4.5 by the addition of 1.0 mol/L NaOH. The culture
was inoculated by aseptically transferring an aliquot of spore sus-
pension to achieve a nal concentration of 105 spores/mL. The
medium was incubated at 26 C with agitation at 250 rpm for
120 h. The culture pH was measured every 24 h and manually
adjusted to pH 4.5 by the addition of 1 M NaOH (Vicente et al.,
2010). All of the experiments were performed in triplicate.
2.3. Lipid extraction
The total lipids were extracted using a microwave-assisted eth-
anol extraction and the solvent extraction method as described by
Folch et al. (1957) and Bligh and Dyer (1959). The microwave-
assisted extraction was conducted with 2.0 g of fungal biomass
(94%, water) in a spherical glass vessel containing 25 mL of ethanol
(96%) heated in a microwave reactor. The microwave reactor
(Model Discover/University-Wave, Cem Corporation) consisted of
a cylindrical internal chamber of 750 mm3 powered with com-
pressed air and equipped with a magnetic stirrer and infrared sen-
sors for temperature control. The operating temperature (50 or
60 C) was controlled automatically by variation in the microwave
power with a maximum of 200 W. The extraction was performed
in three steps of 10, 30 or 60 min each. After each extraction step,
the suspension was ltered and the residual pellet was resus-
pended in the same volume of ethanol for the next step. The
extract containing lipids was recovered and evaporated in a vac-
uum rotary evaporator, and the microbial oil was subsequently
dried at 60 C until it reached a constant weight.
The extraction with the Folchs method was performed with
1.0 g of dried biomass in a glass vessel containing methanol and
chloroform at a 2:1 (v/v) ratio. The extraction was assisted by son-
ication using a UP200S Ultrasonic Processor (Dr. Hielscher GmbH,
Germany) in three steps of 10 min each (pulse mode 0.5 and
100% amplitude). The method of Bligh and Dyer was performed
in a 100-mL Erlenmeyer ask containing 1.0 g of the dried biomass
and 20 mL of a mixture containing chloroform, methanol and
water at a 1:2:0.8 (v/v) ratio and glass beads (1:1, beads to bio-
mass). The mixture was stirred (100 rpm) at room temperature
for 18 h. The total lipids extracted by each technique were mea-
sured gravimetrically after the complete removal of the organic
solvent by evaporation.
2.4. Enzymatic transesterication
Biodiesel synthesis from the single-cell oil of M. circinelloides
was performed in a 50-mL cylindrical glass vessel containing the
microbial oil and anhydrous ethanol at a molar ratio of 1:12. The
reaction used immobilized lipase from Candida antarctica Nov-
ozym 435 as a biocatalyst at concentration of 10% of the total
reaction mixture weight and iso-octane as the reaction solvent.
The reaction was performed at 60 C with constant stirring
(500 rpm) for a maximum period of 72 h (Da Rs et al., 2012).
The biodiesel prepared with palm and jatropha oils under the same
conditions was used as the control.
2.5. Biodiesel purication
After the transesterication reaction, the immobilized lipase
was separated by centrifugation (1520g) and the organic phase
 A.K.F. Carvalho et al. / Bioresource Technology 181 (2015) 4753
was washed twice with one volume of distilled water to remove
any remaining ethanol and glycerol by-product. The residual etha-
nol and water were removed in a vacuum rotary evaporator, and
the fatty acid ethyl esters were analyzed with the international
standard specications for biodiesel.
2.6. Analysis
2.6.1. Scanning electron microscopy (SEM)
High-resolution scanning electron microscopy (LEO Model 1450
VP, ZEISS) was used to analyze the morphology of M. circinelloides
mycelium after the microwave extraction.
2.6.2. Fatty acid composition
An analysis of the fatty acid composition was performed with
capillary gas chromatography (Agilent 6850 Series GC System)
according to AOCS procedure 2-66 (AOCS, 2004). The chromatogra-
phy equipment was equipped with a DB-23 Agilent capillary col-
umn (50% cyanopropyl-methylpolysiloxane) using helium as the
carrier gas at a rate of 1.0 mL/min. The temperatures of the column,
detector and injector were set at 215, 280 and 250 C, respectively.
The volume of the sample injection was 1.0 lL. All of the analyses
were performed in triplicate.
2.6.3. Proton nuclear magnetic resonance spectrometry (1H NMR)
The conversion of microbial oil into FAEEs was analyzed by
NMR in a Mercury 300 MHz Varian spectrometer. FAEEs were dis-
solved in deuterated chloroform (CDCl3) using 0.3% tetramethylsil-
ane (TMS) as the internal standard. The yield of microbial oil
conversion into esters was determined according to the method
proposed by Paiva et al. (2013). This method allows the identica-
tion of ester molecules produced during transesterication, which
causes peaks in the region of 4.054.35 ppm. According to Eq. (1),
the signal of the ethoxyl hydrogen atoms of the ethyl esters split a
quartet.
%EE AC4  8=Addee   100 1 15
where AC4 = area of the component fourth peak (quart), Add+ee = area
of all signals between 4.35 and 4.05, %EE = yield of fatty ethyl esters
(FAEEs).
In this equation, the AC4 was obtained from the integration of
the peak at 4.08 ppm. The area obtained corresponds to 1/8 of
the whole ethoxi-carbon hydrogen area (OCH2), whose signal
appears in the region ranging from 4.05 to 4.20 ppm. The region
near 4.08 ppm is the only region where crossover does not occur,
and this integrated signal can be assigned to ethyl esters.
2.6.4. Mono-, Di- and triglyceride analysis
The glyceride analysis was performed in an Agilent 1200 Series
liquid chromatograph (Agilent Technologies, USA) equipped with
an Evaporative Light Scattering Detector and Gemini C-18 (5 lm,
150  4.6 mm, 110 ) column at 40 C. The mobile phase contained
a mixture of acetonitrile (80%) and methanol (20%) at a ow rate of
1 mL/min for 6 min, 1.5 mL/min until 30 min and 3.0 mL/min until
35 min. All of the samples were dissolved in ethyl acetate-hexane
(1:1, v/v), ltered through 0.22-lm membrane lters (Millipore)
and injected in a volume of 10 lL. All the solvents were of HPLC
grade, and the assays were performed at least in duplicate
(Andrade et al., 2014).
2.6.5. Physicochemical properties
The absolute viscosity of all the oil and biodiesel was deter-
mined with an LVDV-II cone and plate spindle Brookeld viscosim-
eter (Brookeld Viscometers Ltd., England) using a CP 42 cone. A
circulating water bath was used to maintain the temperature of
the samples at 40 C. The shear stress measurements were taken
49
as a function of shear rate, and the dynamic viscosity was
determined as a slope constant. We used 0.5-mL samples, and
the measurements were repeated three times. The density of bio-
diesel was determined with a DMA 35N EX digital densimeter
(Anton Paar, USA) at 20 C. We also used 2.0-mL biodiesel samples,
and the measurements were replicated three times.
The cetane number (CN), degree of unsaturation (DU), long-
chain saturated factor (LCSF), saponication value (SV) and iodine
value (IV) were empirically determined using BiodieselAnalyzer
software (version 1.1) as described by Talebi et al. (2014).
3. Results and discussion
3.1. Production of single-cell oil
The production of single-cell oil by M. circinelloides URM4182
was observed during the cultivation in the medium formulated
with glucose as the main carbon source. The proles of growth,
glucose consumption and lipid production by M. circillenoides are
shown in Fig. 1. The biomass concentration increased rapidly in
the rst 24 h of cultivation and achieved the stationary phase at
72 h. The highest biomass concentration (4.23 g/L), YX/S (0.44 g/g
glucose) and QX (1.06 g/L/d) were obtained at 96 h of cultivation,
and the maximum lipid concentration was 44.0% with a volumetric
lipid production rate (QP) of 0.47 g/L/d (Table 1).
The glucose was slowly consumed and it was not totally con-
sumed (about 30% of the initial concentration remained in the
medium) at the end of cultivation (120 h of growth). This behavior
could be attributed to the insufcient pH control which makes use
of intermittent addition of NaOH 1 mol/L at 24 h intervals.
50
20
Glucose - Biomass (g/L)
40
Lipids (% w/w)
30
10
20
5 10
0 0
0 24 48 72 96 120
Time (h)
Fig. 1. Prole of biomass production (s), glucose concentration (j) and lipids (N)
by M. circinelloides URM 4182 at 26 C and 250 rpm.
Table 1
Biochemical and kinetic parameters of batch cultivation of M. circinelloides URM 4182
using glucose as a carbon source in 96 h.
Parameter Value
Lipid concentration, P (g/L) 1.86 0.01
Biomass, X (g/L) 4.23 0.09
Substrate consumption, S (g/L) 9.82 0.10
Specic yield of lipid, YP/X (g lipid/g biomass) 0.44 0.15
Process product yield, YP/S (g lipid/g substrate) 0.19 0.24
Microbial biomass yield, YX/S (g biomass/g substrate) 0.43 0.05
Volumetric lipid production rate, QP (g/L/day) 0.47 0.06
Volumetric substrate consumption rate, QS (g substrate/L/day) 2.46 0.21
Volumetric biomass production rate, QX (g biomass/L/day) 1.06 0.02
Specic rate of lipid production, qP (g lipid/g biomass/day) 0.11 0.03
Specic rate of substrate consumption, qS (g substrate/g 0.58 0.05
biomass/day) values represent the mean of tripilicate
experiments
50 A.K.F. Carvalho et al. / Bioresource Technology 181 (2015) 4753

Additionally, low oxygen availability in the ask could affect sub-
strate consumption.
However, there are several factors affecting lipid synthesis in
microorganisms including strain, medium and environmental
factors (i.e., pH, temperature, stirring and dissolved oxygen)
(El-Fadaly et al., 2009; Xia et al., 2011). The results achieved in this
study were similar to ndings observed with the cultivation of
M. circillenoides ATCC1216B in a medium formulated with glucose
as the main carbon source (Xia et al., 2011). In another study, bio-
mass and single-cell oil were produced by the growth of a genetically
modied M. circinelloides MU241, which produced 4.2 g/L biomass
concentration and total oil of 22.9% dry mass (Vicente et al., 2010).
The authors reported that the lipid concentration varies considerably
during the rst 48 h of growth (exponential growth phase) and
becomes stabilized when cells reach the stationary phase. The
lipid-accumulating fungi Mortierella isabellina achieved a lipid con-
tent of 67% growing in a medium based on glucose and 50.9% for
medium containing xylose as the main carbon source (Zheng et al.,
2012). The growth of this strain in non-detoxied hydrolyzate of
wheat straw yielded lipid concentrations higher than 39.4%. Most
reports in the literature regarding microbial oil production show
lipid concentration values in the range of 2050%.
3.2. Microbial oil extraction
The effect of different extraction techniques for recovering fatty
acids from M. circinelloides was examined. The content of microbial
oil was assayed by gravimetric estimation. Because the contents of
proteins, pigments and other than non-lipid compounds in the
extract can be considered low, all of the liquid obtained after eth-
anol evaporation can be counted as total microbial oil. The amount
of M. circinelloides oil obtained using different extraction methods
is shown in Table 2.
The total lipid concentration extracted using the ethanol and
microwave method is shown in Fig. 2. The maximum lipid concen-
tration was 41.8% (dry-biomass), which was achieved after three
Table 2
Total lipid concentrations from M. circinelloides URM 4182 as a function of different
extraction methods.
Extraction method Lipids (g) Dried biomass (g)
Microwave-assisted 0.84 0.02 2.02 0.01
ethanol extraction
Folch 0.22 0.01 1.05 0.03
Bligh and Dyer 0.07 0.05 1.03 0.06
50
50C
45 60C
Total lipids ( %, w/w)
consecutive extraction steps at 60 C for 30 min each. However,
the microbial oil obtained under these conditions was only 4%
higher than the lipid attained with extraction steps of 10 min at
the same temperature. Additionally, similar results were veried
for extraction at 50 C.
SEM microphotography of hyphae of M. circinelloides obtained
before and after microwave-assisted lipid extraction with ethanol
is shown in the Supplementary Material (Fig. S1). Most fungi
including M. circinelloides grow as hyphae, which have a cylindrical
form and contain lipids particles and other cytoplasmic organelles.
In this study, the electromagnetic radiation promoted the crushing
of hyphae and we observed small and well-dispersed hyphal frag-
ments (Fig. S1 ab). It is known that absorption of microwave
energy in biological material is dependent on dipolar rotation
and ionic conduction (Datta et al., 2005; Orsat et al., 2005). Mole-
cules such as water and ethanol exhibit strong dipole moments
that can be manipulated in alternating electromagnetic elds,
which results in random collisions of these molecules and leads
to thermal agitation and heating (Datta et al., 2005; Kaufmann
and Christen, 2002). Furthermore, the electric eld created in the
microwave reactor accelerates the oppositely charged ions in dif-
ferent directions and promotes collisions between the particles
to increase the temperature of the material (Kaufmann and
Christen, 2002; Orsat et al., 2005). SEM microphotography also
revealed that some fungal hyphae remained unchanged after
microwave treatment. However, the overall morphology of the pel-
lets (S1-b) suggests that the microwave effectively disrupts cells
for lipid extraction by ethanol.
The comparison of total microbial oil attained by three methods
showed that the microwave led to the best extraction of microbial
oil (Table 2). The cavitation phenomenon produced by ultrasonic
waves disrupts the cells in Folchs method and allows for the
extraction of microbial oil (21.7%, w/w). However, the concentra-
tion was 50% lower than we obtained with the microwave-assisted
method. The extraction with the Bligh and Dyer method was less
efcient compared with the other methods tested (7.0%,w/w).
The glass beads used in this method were ineffective for cell dis-
ruption, which was attributed to the low numbers of bead impacts
against mycelium (high solvent volume to biomass weight ratio).
Iverson et al. (2001) indicated that natural samples containing lipid
concentrations higher than 2% (w/w) could be underestimated by
Total lipids the Bligh and Dyer method due to the low solubility of non-polar
(%, w/w) lipids (triacylglycerols) in solvents that are relatively polar
41.80 0.04 (chloroform, methanol and water). Vicente et al. (2009) reported
a maximal oil concentration of 19.9% (w/w) when dried biomass
21.70 0.06
of M. circinelloides was submitted to three different methods for
7.00 0.05
microbial oil extraction. The methods included mixtures of chloro-
form/methanol, chloroform/methanol/water, and n-hexane. Other
studies described the utilization of solvent for lipid extraction.
However, the modications applied to the different methods (Bligh
and Dyer, and Folch) are unspecied, which makes the evaluation

and comparison of the results difcult. Therefore, ethanol extrac-

40
tion associated with magnetic energy could provide a clean and
35
sustainable method for microbial oil extraction.
30
25 3.3. Single-cell oil composition
20
15 Biodiesel quality is associated with the raw material composi-
tion. Properties including fatty acid composition, chain length,
10
degree of unsaturation, acidity index and iodine number are
5
important parameters determining the potential of the microbial
0 oil for biodiesel production. The characterization of the single-cell
10 30 60
oil from M. circinelloides is shown in Table 3.
Time (min)
Single-cell oil from M. circinelloides revealed fatty acids of
Fig. 2. Single-cell oil obtained by microwave-assisted ethanol extraction from M. carbon chain lengths ranging from 6 to 24. The most abundant
circinelloides URM 4182. fatty acids were oleic acid C18:1; palmitic acid C16:0; and lin-
 A.K.F. Carvalho et al. / Bioresource Technology 181 (2015) 4753 51

Table 3
Properties of single-cell oil from M. circinelloides URM 4182 after 96 h of cultivation in medium containing glucose.

Property Oil
Jatrophaa Palma M. circinelloides (this work)
2
Kinematic viscosity at 40 C (mm /s) 34.5 36.8 36.2
Acidic value (mg KOH/g oil) 0.3 0.3 38.8
Saponication value (mg KOH/g oil) 141 198 203
Iodine value (g I2/100 g oil) 101 98 85
Fatty acids composition (wt.%)
Caproic C6:0 0.3
Caprilic C8:0 0.1
Capric C10:0 0.3
Lauric C12:0 2.1
Miristic C14:0 0.1 1.2 2.1
Pentadecanoic acid C15:0 0.2
Palmitic C16:0 12.9 46.8 22.2
Palmitoleic C16:1 1.0
Margaric C17:0 0.4
Cis-10-heptadecenoic C17:1 0.2
Estearic C18:0 5.6 3.8 7.7
Trans elaidic C18:1 0.3
Oleic C18:1 39.8 37.6 39.3
Trans t-linoleic C18:2 0.2
Linoleic C18:2 40.0 10.5 9.5
Gamma linolenic C18:3 10.7
Linolenic C18:3 0.1
Arachidonic C20:0 0.4
Eicosenoic C20:1 0.7
Behenic C22:0 0.5
Lignoceric C24:0 1.7
Saturated (wt.%) 18.6 51.9 35.8
Monounsaturated (wt.%) 39.8 37.6 41.3
Polyunsaturated (2, 3) (wt.%) 40.2 10.5 20.5
Degree of unsaturation (%) 120.2 58.6 82.5
a
Carvalho et al. (2013).

oleic acid C18:2. Other fatty acids (C8:0, C10:0, C12:0, C14:0,
C16:0, C17:0, C18:0, C20:0, C22:1 and C24) represented less than
8% of the total fatty acid content. The high amount of saturated
(35.8 wt.%) and monounsaturated fatty acids (41.3 wt.%), such as
C16:0 and C18:1, shows the potential use of this oil for biodiesel
production and other industrial applications. This prole has sim-
ilar characteristics to vegetable oils conventionally employed for
biodiesel production (Carvalho et al., 2013). Linoleic acid C18:2
and c-linolenic acid (GLA) C18:3 (10.7 wt.%) are the polyunsatu-
rated fatty acids (PUFAs) with the highest concentrations (Table 3).
These compounds are highly desirable because they have applica-
tions for food, pharmaceutical and cosmetic formulation. Vicente
et al. (2009) reported oil production from glucose by M. circinello-
ides with high concentrations of palmitic acid (20.0 wt.%), oleic acid
C18:1 (37.0 wt.%) and c-linolenic acid C18:3 (18.5 wt.%). In another
study of the growth of Aspergillus terreus on hydrocarbon substrate,
fatty acids with chain lengths of C14C18 were predominant in the
lipid prole and the PUFAs (C15:4, C17:4, C19:4, C32:3, and C33:4)
were approximately 9% (wt.%) (Kumar et al., 2010).
Some other properties of the oil used for biodiesel production
have also signicant inuence on the biofuel characteristics. The
iodine value (85 g I2/100 g oil) of the microbial oil produced by
M. circinelloides indicated a high degree of saponiable lipids,
including triglycerides, diglycerides, monoglycerides and free fatty
acids. This value is lower than the normal established value. Fur-
thermore, we observed a high degree of unsaturation (82.46%) that
could generate methyl and ethyl esters with low thermal-oxidative
stability. This characteristic is desirable because there is an inverse
relationship between the oxidative stability of the lipids and the
hydrodynamic property (uidity) of the biofuels produced from
certain oil. Thus, the high degree of unsaturation of the biosynthe-
sized oil indicates that it is suitable for the production of biodiesel
for combustion at low temperature because it has a low risk of
solidication. In addition to the melting point, the number of
unsaturated carbonic chains of the lipids used for biofuel produc-
tion affects the cetane number, combustion heat (measurement
of energetic power) and boiling point of biodiesel (Canakci et al.,
1999; Vicente et al., 2009).
Polyunsaturated fatty acids (PUFAs) with four or more double
bonds are usually undesirable for biodiesel production and were
not identied in the single-cell oil obtained from M. circinelloides.
A comparison of the microbial oil from M. circinelloides and palm
oil shows that both oils have similar oleic acid and monounsatu-
rated fatty acid compositions (Table 3). In general, oil from la-
mentous fungi differs from vegetable oil in the high
concentration of PUFAs, which are compounds that are especially
important for obtaining intermediate molecules for chemical,
pharmaceutical and food industries (Khot et al., 2012; Vicente
et al., 2009). We also observed that there was approximately 50%
less polyunsaturated lipids in single-cell oil from M. circinelloides
than in the non-edible jatropha oil, which is another important
source for biodiesel production (Table 3).
3.4. Biodiesel production
The single-cell oil obtained from M. circinelloides was subjected
to transesterication using ethanol as an acyl acceptor and Candida
antarctica lipase B immobilized on macroporous acrylic resin as the
catalyst following methodology previously established for micro-
bial oil (Da Rs et al., 2012).The conversion of the microbial oil
and purity of the biodiesel was assessed by nuclear magnetic res-
onance spectroscopy (1H NMR) and liquid chromatography (HPLC)
analysis.
NMR is extensively used to monitor the transesterication pro-
cess and quality of biodiesel. This analysis is based on the variation
of glycerol methylene and ester ethoxy hydrogens in the region of
52 A.K.F. Carvalho et al. / Bioresource Technology 181 (2015) 4753

4.054.35 ppm during the transesterication reaction (Paiva et al., and microbial oils. Vicente et al. (2009) reported the extraction
2013). The 1H NMR spectrum of single-cell oil and fatty acid ethyl and conversion of oil from M. circinelloides into fatty acid methyl
esters (FAEE) of M. circinelloides are shown in Supplementary Mate- esters (FAMEs) by a homogeneous acid catalyst (BF3; HCl and
rial (Fig. S2). The spectrum shows a peak at 4.08 ppm, correspond- H2SO4). The authors achieved yields ranging from 91.5% to 98.0%.
ing to methylene protons of the ethanolic fraction of ethyl esters. The authors also described a one-step (direct) production of bio-
Table 4 shows the areas of the peak corresponding to ethyl esters diesel from biomass of M. circinelloides using the same conditions
and the percentage conversion of the single-cell oil. Botn experi- for producing biodiesel with yields higher than 99.0% (Vicente
ments reached yields of around 93% after 72 h reaction. The et al., 2009). In a previous work, B. cepacia lipase immobilized on
absence of peaks (multiples) in the 4.20 ppm region of the biodie- SiO2-PVA was used at a xed proportion of 500 units per gram of
sel spectrum that correspond to the protons of the 1 and 3 carbon palm and jatropha oil to catalyze biodiesel synthesis. This
of glycerol in the triglyceride molecules conrms the high conver- approach resulted in FAEEs yields of 99.6 and 98.5%, respectively
sion of the microbial oil. (Carvalho et al., 2013).
The chromatography analysis conrmed this high rate of The biodiesel quality is dependent on the fatty acid prole of
conversion 93% 0.4 of the single-cell oil of M. circinelloides into the oil used as feedstock for its production. In addition to the
biodiesel (Table 5). The amount of monoglycerides and diglyce- molecular characteristics of the oils, properties such as cetane
rides found were 4.2% and 2.8%, respectively. The ASTM standard number (CN), kinematic viscosity, density and cold lter plugging
for biodiesel (ASTM D6751) establishes values up to 0.5% for mono point (CFPP) are parameters widely used to characterize biodiesel.
and diglycerides. Generally, the presence of unreacted glycerides The CN is a measurement of the combustion quality during igni-
reduces the quality of the nal product because these compounds tion of biodiesel and depends on the distribution of saturated and
increase the turbidity and viscosity of the product. The presence of unsaturated fatty acids or esters. The ignition quality is associated
the glycerides and lower conversion of the oil could be associated with the interval of ignition. A fuel with high CN number has a
with the high concentration of C18:3, which is a polyunsaturated short ignition interval and starts to burn shortly after it is injected
fatty acid with a long carbon chain. The catalyst (immobilized into an engine. In this study, CN was estimated at 49.2, 59.3 and
lipase) is more efcient for the conversion of saturated fatty acids 54.2 for blends of ethyl esters from jatropha, palm and M. circinello-
with short carbon chains (Christopher et al., 2014). ides oil, respectively (Table 5). These values are in compliance with
Further removal of these impurities can assure the standard the standards for biodiesel (ASTM D6751), which recommend a
quality of the biodiesel from M. circinelloides. Furthermore, the minimum CN of 47.The European minimum CN number is 51,
low concentration of these unreacted glycerides and the absence and all of the biodiesel complies with the specications except
of triglycerides in biodiesel from M. circinelloides show the ef- for biodiesel from palm oil. Generally, high CN values are observed
ciency of conversion of triglycerides into ethyl esters promoted for saturated fatty esters and increase with chain length
by the immobilized lipase. (Varatharajan and Cheralathan, 2012). CN values greater than the
The characteristics of the FAEEs obtained from M. circinelloides minimum specied by the engine manufacturer do not necessarily
were compared to FAEEs produced from palm and jatropha oil increase the engine performance. This value is associated not only
(Table 5), which are raw materials used extensively by the biodie- with the molecular composition of lipid used for biofuel produc-
sel industry in Brazil. A comparison of the biodiesel obtained under tion but also with the engine design and characteristics (Knothe
the same conditions from jatropha and palm oil shows yields et al., 2005).
slightly higher than the yields obtained using the single-cell oil In addition to cetane number, viscosity is another parameter
from M. circinelloides. These results are comparable to data that affects biodiesel quality. The kinematic viscosity is dependent
reported in the literature for biodiesel produced from vegetable on the degree of unsaturation and chain length of the fatty acid
blends or alcohol moiety that compounds the biodiesel. Viscosity
increases with molecular weight but decreases with increasing
Table 4 level of unsaturation and temperature (Knothe and Steidley,
Area of the integration peaks and the conversion percentage of single-cell oil of M. 2005). A higher viscosity results in low volatility and poor atomiza-
circinelloides to ethyl esters determined by 1H NMR. tion of the fuel during the injection, which promotes an incomplete
Transesterication reaction Area AC4 Area Add+ee Yield (%)
burning and carbon deposits in the injector and combustion cham-
ber (Knothe et al., 2005; Ramos et al., 2009). In the present study,
T1 0.11 0.94 93.5
T2 0.10 0.86 92.9
the kinematic viscosity obtained for biodiesel from M. circinelloides
was slightly higher than the limit established by the standard (1.9
6.0 mm2/s). The biodiesel samples from jatropha and palm are
within the standard range (Table 4).
The cold lter plugging point (CFPP) is the lowest temperature
Table 5 at which a given volume of fuel passes through a standard lter in
Biodiesel characteristics obtained from transesterication of vegetable (jatropha and a specied time with specic cooling conditions. Similar to the CN,
palm) and single-cell (M. circinelloides) oils using ethanol as acyl acceptor.
the CFPP of biodiesel mainly depends on the saturated fatty acid
Jatropha Palm SCO composition (Knothe and Steidley, 2005; Ramos et al., 2009). This
curcas value is especially important in cold temperature countries where
Ester content (%)a 96.1 0.4 98.0 0.6 93.0 0.4 a high CFPP could clog engine lters. The CFPP for biodiesel from M.
Monoglycerides (%) 3.2 0.2 2.0 0.1 4.2 0.2 circinelloides was estimated at 3.9 C and is similar to values
Diglycerides (%) 0.8 0.1 0 2.8 0.1
observed for biodiesel from jatropha and palm oil (Table 4). These
Triglycerides (%) 0 0 0
Density at 20 C (kg/m3) 883 0.5 874 1.6 878 1.9 values are in compliance with the Brazilian biodiesel standard that
Cetane number 49.2 59.3 54.2 establishes a maximum CFPP of 5.0 C for colder regions of the
Kinematic viscosity at 40 C (mm2/s) 4.8 0.5 5.5 0.3 6.2 0.2 country (ANP). The standard specication of CFPP is dependent
Cold lter plugging point (CFPP, C) 2.4 4.2 3.9
on the season and location. In other countries using UNE-EN
Degree of unsaturation (%) 120.2 58.6 82.5
14214 or ASTM D6751, various temperature limits are specied
a
Total ethyl ester formed with respect to the initial oil mass in the reaction for different times of the year depending on climate conditions
medium, i.e., YGlobal = [(biodiesel mass)/(oil mass)]  100. (Knothe, 2006).
 A.K.F. Carvalho et al. / Bioresource Technology 181 (2015) 4753
4. Conclusions
Single-cell oil from M. circinelloides was analyzed as a potential
feedstock for biodiesel production. The total oil obtained was 44%
of dried biomass after 96 h of cultivation. Microwave-assisted eth-
anol extraction is a fast and sustainable method for recovering high
lipid concentrations from fungi. The lipid prole of M. circinelloides
showed high concentrations of saturated and monounsaturated
fatty acids, predominantly C16:0 and C18:1. The enzymatic transe-
sterication of the single-cell oil with ethanol achieved yields of
ethyl esters of 93%. The transesterication of the single-cell oil
from M. circinelloides is a suitable approach to completely fulll
the standard specications for renewable diesel.
Acknowledgements
The authors gratefully acknowledge the nancial support of
Coordenao de Aperfeioamento de Pessoal de Nvel Superior
(CAPES) and Conselho Nacional de Desenvolvimento Cientco e
Tecnolgico (CNPq). Juan D. Rivaldi would especially like to thank
the support of CAPES (Process PNPD 02565/09-9).
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.2014.12.
110.
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