Journal of Sustainable Bioenergy Systems, 2013, 3, 224-233
http://dx.doi.org/10.4236/jsbs.2013.33031 Published Online September 2013 (http://www.scirp.org/journal/jsbs)
Biodiesel Production fro m Spirulina-Platensis Microalgae
by In-Situ Transesterification Pr ocess
H. I. El-Shimi1, Nahed K. Attia2*, S. T. El-Sheltawy1, G. I. El-Diwani2
1Chemical Engineering Department, Faculty of Engineering, Cairo University, Giza, Egypt
2Chemical Engineering and Pilot Plant Department, National Research Center, Dokki, Egypt
Email: firstname.lastname@example.org, *email@example.com
Received June 8, 2013; revised July 10, 2013; accepted July 30, 2013
Copyright © 2013 H. I. El-Shimi et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This research investigates the effect of reaction variables that strongly affect the cost of biodiesel production from
non-edible Spirulina-Platensis microalgae lipids, and use the acid-catalyzed in situ transesteriﬁcation process. Experi-
ments were designed to determine how variations in volume of reacting methanol, the concentration of an acid catalyst,
time, temperature and stirring affected the biodiesel yield. The total lipid content of Spirulina-Platensis microalgae was
obtained to be 0.1095 g/g biomass. The weight of the by-product glycerol obtained was used to predict the percentage
yield conversion of microalgae oil biodiesel. Best results (84.7%), a yield of fatty acid methyl ester (FAME), were ob-
tained at 100% (wt./wt.oil) catalyst concentration, 80 ml methanol volumes, 8 h reaction time and 65˚C reaction tem-
perature with continuous stirring at 650 rpm. Properties of the produced biodiesel were measured according to EN
Keywords: Biodiesel; Spirulina-Platensis; Microalgae; In-Situ Transesterification
Energy today is the most important resources for man-
kind and its sustainable development, due to the energy
crisis which becomes one of the global problems con-
fronting the world . Major energy resources come
from fuels, due to their energy content with significant
amounts. Nowadays, there is a strong dependence of our
life on fossil fuels such as petrol oil, coal and natural gas,
since more than 80% of the world’s energy needs are
from fossil fuels, whatever, in the industrial production
sector, domestic uses or in the transportation sector. The
problem also is that the population growth is not covered
by domestic crude oil production and its derivatives .
In addition, the formation of fossil fuels requires millions
of years, hence the petrol fuels are non-renewable. Also,
change of the crude oil prices leads to global and interna-
tional conflicts especially in the developing countries.
Renewable energy is considered as one of the most impor-
tant resources in many countries around the world, which
accounts for about 10% of the world’s energy consump-
tion and can be converted to other usable forms of energy
like biofuels . Liquid biofuels have become a green
important alternative fuel that offers several advantages
including its renewability, high energy content and low
emission profile of carbon dioxide .
Liquid biofuels are classified into three generations
based on the feedstocks and production technology .
First generation liquid biofuels—bioethanol and bio-
diesel—were produced from food crops such as corn,
sugarcane and vegetable oils. Since the food crops are
used in the fuel production, first generation liquid biofu-
els were limited to conflicting with the food supply and
increasing the food crop prices. This has paved the way
for second generation liquid biofuels, which were pro-
duced, using waste cooking oil, non-edible plant seed oil,
waste vegetable oil and animal fats [5,6]. Although sec-
ond generation liquid biofuels overcame the problems
faced by their first generation counterparts, increasing
the fuel consumption and creating a challenge for the
supply with consistent feedstock, this difficulty led to the
development of third generation liquid biofuels like algae
Biodiesel (fatty acid alkyl esters, FAAE) is a green al-
ternative liquid diesel fuel derived from vegetable oils or
lipids by the reaction with alcohol in the presence of a
catalyst. Biodiesel is used today as the basis for a clean
substitute for petrol-diesel without any modification in
opyright © 2013 SciRes. JSBS
H. I. EL-SHIMI ET AL. 225
diesel engines . Biodiesel is environmentally-friendly,
non-toxic and biodegradable fuel, which can be made
from any vegetable oils (edible or non-edible), animal
fats or special strains of microalgae .
Microalgae has been considered recently as a prom-
ising biomass feedstock with great potential for biodiesel
production  because they reproduce themselves every
few days (2 or 3 weeks), yield oil exceeding 10x the
yield of the best oilseed crops, reduce emissions of a
major greenhouse gas (1 kg of algal biomass requiring
about 1.8 kg of CO2) and can be obtained from wastewa-
ter (1 m3 of wastewater is required to produce 800 g of
dry algae). In addition, microalgae as a fuel source does
not conflict with the food crisis, since it is not the main
food source. The production of biodiesel using microal-
gae biomass as a possible feedstock has been described
by Chisti Y. .
The production of biodiesel from microalgae oil by
transesterification process has previously been demon-
strated in the literature using the conventional methods [12,
13], and the process usually uses pre-extracted oil as raw
material, which is usually produced  by mechanical
pressing followed by solvent extraction to extract the
remaining oil, and then its conversion to FAAE and
glycerol. The transesteriﬁcation reaction can be catalyzed
by alkali [15-17], acidic , or enzymes [19-23]. The
use of the alkaline catalysed transesteriﬁcation technol-
ogy would not be suitable for biodiesel production from
microalgae oil; because of the high FFA content of mi-
croalgae lipids. This is because the use of alkaline cata-
lysts with high FFA containing oils would result in soap
formation [24,25] and difﬁculties in the biodiesel separa-
tion and puriﬁcation downstream. The use of sulphuric
acid, as reaction catalyst, has been considered as micro-
algae lipid transesteriﬁcation, due to its insensitivity to
the FFA content of this oil feedstock, as the transes-
teriﬁcation and esteriﬁcation reactions of biodiesel pro-
duction are facilitated via acidic catalysis , however,
acidic transesterification process is limited due to the
water formation during the esterification reaction, high
alcohol-to-oil ratio (about 40:1), and large amounts (5%
to 25%) of catalysts may be required . Also the use
of enzymes as a transesterification catalyst is still under
study. The biodiesel production from microalgae on an
industrial scale still faces problems, mainly due to the
high costs associated with the present biomass produc-
tion and fuel conversion routes .
One of the alternatives to produce biodiesel from mi-
croalgae lipids is “in-situ transesteriﬁcation” or “reactive
extraction” process [14,27]. This process combines the
steps of lipid (oil) extraction and transesteriﬁcation to
produce biodiesel. Integration of these stages could
minimize biodiesel production cost , since the use of
reagents and solvents is reduced and the analysis is easier
and not expensive. The method involves the simultane-
ous addition of the acid catalyst and pure methanol to
microalgal biomass (generally in the form of dried
powder). The methanol extracts the lipids from the mi-
croalgal biomass and, catalyzed by the acid, concurrently
transesteriﬁes the extracted lipids to produce fatty acid
methyl esters [29,30].
The method was ﬁrst demonstrated by Harrington and
D’Arcy-Evans  with sunﬂower seeds as feedstock,
using the in situ method, and these authors achieved an
increase in biodiesel yields up to 20% compared to the
conventional process. This improvement in the biodiesel
yields was considered by these authors to be attributable
to the improved accessibility of the oil in the biomass by
the acidic medium. The in situ transesteriﬁcation of mac-
erated sunﬂower seeds was also studied by Siler-
Marinkovic et al.  who investigated two temperature
levels of 30˚C and 64.5˚C and a range of test reaction
conditions: the alcohol (methanol) to oil molar ratio var-
ied from 100:1 to 300:1, and the sulphuric acid catalysts
concentration ranged from 16% to 100% (on the basis of
the oil) and a reaction time of 1 - 4 h. Under the condi-
tions studied, the best FAME yields (98.2%) based on the
oil content of the sunﬂower seeds were obtained at a mo-
lar ratio of methanol to oil of 300:1, an acid catalyst
concentration of 100% and a reaction time of 1 h.
The main objective of the present work is to apply the
biodiesel production technology using an acid catalyst to
the in-situ transesteriﬁcation of microalgae (Spirulina-
Platensis), where the main reaction variables that
strongly affect the cost of this process were studied.
These variables are: 1) the catalyst concentration (the
larger the catalyst concentration, the more the material
costs input); 2) the reacting alcohol volume (also, the
more the alcohol volume, the more the material costs
input); 3) the temperature (increasing of temperature, and
increasing the process of energy requirement); 4) the
reaction time (the larger the reaction time, the lower the
product amounts yielded, and lower the product profit);
and 5) process stirring (main energy requirement for the
reaction agitation). This investigation has been carried
out to provide information on the optimum operating
conditions that give the best yield while also having the
lowest material and energy requirements, and conse-
quently lowest process costs, since the use of the in situ
transesteriﬁcation process as a proper biodiesel produc-
tion technique is mainly driven by its possible applica-
tion with relatively low cost.
2. Materials and Methods
Spirulina-Platensis microalgae were supplied from the
Microbiology Department, Soils Water and Environment
Res. Inst., Agriculture Research Center (ARC), Giza,
Egypt. This microalgae strain was collected from three
Copyright © 2013 SciRes. JSBS
H. I. EL-SHIMI ET AL.
weeks old. The culture media used was the same of Zar-
rouk’s medium . The cultivation of Spirulina-Plat-
ensis was in mini-tanks with dimensions similar to that
used in Ref. . The cultivation was carried out at 30˚C,
3.5 klx of illuminance provided by fluorescent lamps and
pH of 8.5 ± 0.5. At the end of the culture cycle, algal
suspensions were homogenized (Homogenizer Wisetis
HG-15D) for 10 minutes at 1800 rpm; to disrupt the cells
and ease the oil extraction, and filtered through Centri-
fuge separator (Beckman CS-6 Centrifuge 3500 rpm,
Germany) and then dried to a constant weight using solar
drying beds and storedat 18˚C until use.
Sulphuric acid of 98% purity is used in this study as a
catalyst in the transesterification process. Methanol
(99.9% purity) was used as the reacting alcohol in this
Microalgae oil was extracted using the Soxtherm extrac-
tion system described by Jie Sheng et al. . After re-
extraction with methanol as a solvent, followed by frac-
tional distillation to recover the microalgae oil, the ex-
tracted oil was weighed to determine the total lipid con-
tent per dry algal biomass, and then analyzed; to charac-
terize the properties of Spirulina-Platensis oil.
Variable sulphuric acid concentrations (0.0046, 0.0077,
0.0154 & 0.0308 mol), were used throughout this study.
The acid-methanol solution was prepared freshly by
mixing predetermined amounts of sulphuric acid and
methanol. H2SO4 was dissolved with continuous stirring
on a magnetic stirrer for 5 min. The solution was pre-
pared freshly in order to maintain the catalyst activity.
Dried microalgae of 15 gm was added carefully to
catalyst/alcohol mixture and blended on low setting for
several minutes. At this point, the simultaneous extrac-
tion and transesterification reaction has been initiated;
where the catalyst/alcohol solution attacked the triglyc-
eride (oil) in the microalgae strain and cleaved off a fatty
The vessels containing the reaction mixtures were then
heated and maintained at the temperatures of interest for
speciﬁed periods. The major in situ transesteriﬁcation
reaction and product puriﬁcation steps used are shown in
2.2.1. Settling and Separati ng
After the transesteriﬁcation step “Figure 1(a)”, the warm
reaction mixture was allowed to cool for 20 min. The
reaction mixture was ﬁltered and the residues are washed
three times by re-suspension in methanol (45 ml) for 15
min to recover any traces of FAME product left in the
residues “Figure 1(b)”. Water (60 ml) was added to the
ﬁltrate, to facilitate the separation of the hydrophilic
components of the extract, and then poured into a
500-mL separating funnel “Figure 1(c)” and the reaction
vessel was allowed to stand for 4 h to enable its contents
to settle. Further extraction of the FAME product was
achieved by extracting three times for 15 min using 60
ml of hexane “Figure 1(c)”, which resulted in generation
of two layers: hydrophobic layer (hexane, FAME and
glycerides), and hydrophilic layer (water, glycerol and
The reaction was demonstrated to be successful by
observing the glycerin settling in the bottom soon after
stopping mixing of the reactants. The top of the mixture
looked lighter, and a darker layer was formed at the
bottom. When the product has fully settled, two distinct
layers were separated. These two layers are alkyl esters
(biodiesel) and glycerin. The biodiesel on top looked as a
clear, lighter in color, thin, and slippery to the touch. The
glycerin settled to the bottom looked clear, darker amber
color, thick, and sticky to the touch. Most of the settling
occurred within the first hour. Once the glycerol and
biodiesel phases have been separated, the bottom layer
which contains glycerol, trace water, catalyst, and excess
methanol was drawn into a pre-weighted beaker and
dissolved in pure water; to purify the glycerol layer, and
then subjected to a flash evaporation process “Figure
1(d)”, in which excess alcohol and water are removed.
The recovered alcohol was recycled and reused. Now,
the layer contains only the by-product glycerol and the
catalyst, therefore the weight of pure glycerol can be
detected by the well-known catalyst weight. This pro-
cedure was performed in each experiment of the work,
since we took a 15 g of microalgae biomass in each
experiment, which expected to contain lipids of 1.6425 g,
and based on just 60% reaction conversion, around 0.99
g glycerol will be obtained and can be weighted; using
four digits balance.
2.2.2. Meth yl Ester (Biodiesel) Wash
The top layer in the separation funnel is the produced
biodiesel. This biodiesel layer was washed with water
“Figure 1(e)” and filtered into a clean, dry side-arm flask;
to evaporate the methanol and the hexane using a frac-
tional distillation apparatus “Figure 1(f)”. The amount of
collected biodiesel is difficult to be measured; since the
unreacted glycerides are mixed with it, so the yield of the
FAME can be calculated using the balanced equation of
the transesterification reaction and then compared with
the microalgae oil to monitor the extent of the conversion.
With the forward reaction resulting in FAME production
and the process is near to completion, the weight of the
purified glycerol as a co-product (after the removal of the
water and excess alcohol and omitting the weight of the
catalyst used) is expected to increase until a constant
value, signifying an equilibrium conversion of the micro-
algae lipids to the methyl esters.
Copyright © 2013 SciRes. JSBS
H. I. EL-SHIMI ET AL.
Copyright © 2013 SciRes. JSBS
Figure 1. Block diagram of the in situ transesteriﬁcation steps used for biodiesel production from Spirulina-Platensis biomass.
2.3. Analytical Method
Fatty acids composition of the extracted algae oil was
determined using gas chromatographic analysis of the oil
ethyl esters. Modification of the oil to its ethyl esters was
made using 2% H2SO4 as catalyst in the presence of dry
ethyl alcohol in excess. The chromatographic analysis
was made using Hewlett Packard Model 6890 Chro-
matograph. A capillary column 30 m length and 530 μm
inner diameter, packed with Apiezon® was used. De-
tector temperature, injection temperature and the column
temperature were 280˚C, 300˚C and 100˚C to 240˚C at
2.4. Variables Affecting the in situ
2.4.1. Effect of Alcohol Volume and Temperature
Spirulina-platnsis powder (15 g) was mixed with vari-
ous methanol volumes (40.0, 60.0, 80.0 and 100.0 ml)
containing 2.2 ml of sulphuric acid (as the optimum
catalyst concentration) in screwed cap reaction vessels as
described before. A minimum volume of 40.0 ml metha-
nol was selected since it was the suitable amount that
facilitated a complete submersion of 15 g of the micro-
algae powder. The experiment involved heating the re-
action mixtures in flat bottom round flask for 8 h, with
each trial at one of four different temperatures (27˚C,
40˚C, 50˚C and 65˚C) with continuous stirring using a
hot plate with a magnetic stirrer. The respective FAME
products and the co-product glycerol at different in-
vestigated variable levels were obtained and their
2.4.2. Effect of Catalyst Concentration
Spirulina-platensis powder (15 g) was mixed with 80 ml
methanol containing different moles of sulphuric acid
(0.0046, 0.0077, 0.0154 and 0.0308 mol) whose relate to
(30%, 50%, 100% and 200% respectively) acid catalyst
concentration (on the basis of the microalgae oil content
mass), this was carried out at 65˚C for 8 h. Also the in
situ transesterification reaction was performed at the
same conditions without catalyst; to provide a greater
insight on the effect of the catalyst presence in the tran-
2.4.3. Effect of Reaction Time
At each of the four temperature levels, the in situ tran-
sesteriﬁcation of 15 g microalgae biomass was repeated
in duplicate with reaction times of 2, 4, 8 and 10 h with
80 ml methanol containing 2.2 ml sulphuric acid.
This was carried out to provide a greater insight on the
progression of the transesteriﬁcation process with time
with respect to the various investigated reaction tem-
peratures. The puriﬁcation of the glycerol co-product and
its weight determination was carried out as described
2.4.4. Effect of Stirring
To investigate the effect of stirring, the reaction vessels
used for the in situ transesteriﬁcation process were run
with and without stirring for comparison. The reaction
stirring was carried out using a magnetic stirrer system
with a rotation speed of 650 rpm kept constant through-
out the duration for the reaction. This speed was used
since it was observed to facilitate a complete suspension
of the particles in the reaction vessels. For each treatment,
transesteriﬁcation was carried out as before using 15 g
biomass with 80 ml of methanol containing 0.04 mol
sulphuric acid with a reaction time of 8 h and a tempera-
ture of 65˚C. The reaction co-product (glycerol) was
H. I. EL-SHIMI ET AL.
puriﬁed and its weight was determined.
3. Results and Discussion
3.1. Lipid Content and Properties of Pure
According to the culture conditions used in this study,
the Spirulina platensis samples were determined to have
a total lipid content of 10.95% wt. of Spirulina platensis
biomass. The biomass oil content of the used microalgae
strain is highly dependent on the speciﬁc growth condi-
tions not only inﬂuenced by the microalgae specie .
The microalgae culture conditions, nutrients and light
intensity can be optimized to increase the oil content of
the biomass, and hence increases in the biodiesel produc-
The properties (such as density, viscosity and acid
value) of the extracted oil, which were used to character-
ize the reacting oil at the start of the transesteriﬁcation
reaction, were determined. The results for the percentage
principal fatty acids of the extracted microalgae oil, as
detected via GC analysis of the resulting FAME mixture,
are shown in “Table 1”. This data were used to deter-
mine the average molecular mass of the Spirulina-plat-
ensis oil. In these results, fatty acids detected only in
trace amounts (<1%) were not included. Microalgae oil is
composed of different fatty acids, so their respective
contributions to the overall molecular mass of the micro-
algae lipid is investigated (as illustrated in the last col-
umn of “Table 1”); to estimate the average molecular
mass of the constituent lipid fatty acids (MMFA).
Table 1. Calculations of the molecular mass of Spirulina-
Fatty acid Molecular Mass
(g/mol) (MMFA) % in sample
C14:0 242 22.6718 54.8658
C16:0 256 49.5806 126.9263
C16:1 254 2.7491 6.9829
C18:0 284 5.5645 15.8034
C18:1 282 2.2435 6.3266
C18:2 280 5.0347 14.0971
C18:3 278 7.4033 20.5812
C20:0 312 1.0601 3.3076
C20:1 310 3.6921 11.4456
Average Molecular Mass of Constituent Fatty Acids
Since the microalgae oil has quite big molecules with a
spinal of glycerol on which are bond three fatty acid rests,
by the transesterification the fatty acid rests are removed
from the glycerol and each is bond with methanol, and
three molecules of water are condensed, the average mo-
lecular mass of the microalgae oil (MMoil) can be calcu-
lated using “Equation (1)”.
where, MMglycerol and MMOH, H represent the molecular
masses of glycerol and OH group and a hydrogen atom,
respectively. The average molecular weight of the Spiru-
lina-platensis oil was calculated to be 845.19 g/mol.
To calculate the molecular mass of the FAME (bio-
diesel); as the reaction yield calculations are based on it.
The reaction yield is calculated from “Equation (2)”. The
amount of biodiesel can be determined from the stio-
chiometric equation of the transesterification reaction, by
knowing the weight of glycerol. And as mentioned be-
fore that we could determine the weight of the reaction
co-product “glycerol” from the experimental work.
Algae Biodiesel Yield %WeightofMicroalgae Oil
The FAME molecular mass can be calculated as simi-
lar to microalgae oil, but it can be calculated according to
the chemical reaction of the transesterification process
shown in “Figure 2”. The FAME chemical formula is
increased over that of the average molecular mass of
constituent fatty acids, so the molecular weight of bio-
diesel can be calculated from “Equation (3)”:
Since the molecular mass of substituted CH3 group is
15. Therefore, the average molecular weight of the bio-
diesel is 284 g/mol.
The acid value of the microalgae oil was determined to
be 37.4 mg KOH/g Spirulina-platensis oil. Using the
estimated molecular mass of 269.065 for the constituent
fatty acids, the FFA content of the microalgae oil was
determined to be 18.7% (on the basis of the oil weight).
Due to the high FFA content (>2% w/w) of the microal-
gae oil, the choice of acidic over alkaline catalysts for the
Figure 2. Overall transesterification reaction; where, R1,
R2, R3 are three fatty acids.
Copyright © 2013 SciRes. JSBS
H. I. EL-SHIMI ET AL. 229
in situ transesteriﬁcation process is justiﬁed.
3.2. Effect of Alcohol Volume
One of the most important variables affecting the yield of
methyl esters is the molar ratio of alcohol to triglycerides.
The stoichiometric ratio for transesterification requires
three moles of alcohol and one mole of triglycerides to
yield three moles of fatty acid methyl esters and one
mole of glycerol. However, transesterification is an equi-
librium reaction in which an excess of alcohol is required
to drive the reaction to the right [37,38].
According to the average molecular mass of the
Spirulina-Platensis oil, that was determined in Section
3.1, the methanol volumes investigated in this study rep-
resent a reacting alcohol to oil molar ratio ranges of
1857:1 - 4643:1 as shown in Table 2 (calculated accord-
ing to methanol density of 0.7918 g/cm3). This range
includes and exceeds that of a similar investigation of the
in situ transesteriﬁcation of sunﬂower oil by Siler-
Marinkovic and Tomasevic , and that of Chlorella oil
by E.A.Ehimen .
The percentage yield of the produced FAME was cal-
culated based on the total amount of co-product glycerol
obtained, concerning experimental and analytical error to
be ±5% for the investigated reacting methanol volumes
“Table 2”, using ﬁxed reaction time of 8 h, temperature
of 65˚C, and a ﬁxed acidic catalyst molar concentration
(0.0154 mol sulphuric acid) at constant stirring rate of
The obtained results are presented in “Figure 3” indi-
cate an improvement of the microalgae oil conversion to
FAME with increasing alcohol volume, with the lowest
FAME equilibrium conversions observed with the react-
ing molar ratios of the methanol to oil at 1857:1 (metha-
nol volume of 40 ml) for all the conditions studied.
However, with the use of alcohol volumes over 80 ml (i.e.
a reacting molar ratio of alcohol to microalgae oil greater
than 3714:1) for the in situ transesteriﬁcation of 15 g
microalgae biomass, no signiﬁcant trends were observed
for the FAME yields.
3.3. Effect of Catalyst Concentration
One of the most important variables affecting the yield of
FAME is the concentration of the acid catalyst. These
Table 2. Effect of alcohol volume on biodiesel yield.
Alcohol vol. ml Molar ratio (x:1) Yield %
40 1857.07 73.2
60 2785.61 81.79
80 3714.16 84.7
100 4642.69 84.7
Figure 3. Effect of alcohol volume on biodiesel yield (at
65˚C for 8 hr, stirring of 650 rpm and H2SO4 100% wt./wt.
results agree with the methanolysis with 100% (wt./wt.
of oil) using sulphuric acid catalyst resulted in successful
conversion of Chlorella oil giving the best yields and
viscosities of the esters by E.A. Ehimen et al. .
In this research the in situ transesterification process
was studied at four catalyst loadings (30%, 50%, 100%
and 200% H2SO4 wt./wt. algae oil content) as illustrated
in “Table 3”. Higher yields of 84.716% (with ±5% ana-
lytical error) were reported with 100% H2SO4 (wt./wt. oil)
at 65˚C for 8 hr using methanol-to-oil molar ratio of
3714:1, with further increase in catalyst concentration the
conversion efficiency more or less remains the same. The
effect of catalyst concentration on the yield of fatty acids
methyl esters is presented in “Figure 4”.
3.4. Effect of Reaction Time and Temperature
To investigate the inﬂuence of reaction time and tem-
perature, a methanol volume of 80 ml was used since it
was found (Section 3.2.) that no appreciable differences
in the equilibrium FAME conversion were obtained with
the use of higher alcohol volumes. Reactions were car-
ried out at different temperatures of 27˚C up to 65˚C as
shown in “Table 4”, using methanol-to-oil molar ratio of
3714:1, catalyst concentration of 100% (wt. /wt. oil) and
constant stirring rate of 650 rpm.
The progress of the microalgae oil to biodiesel conver-
sion process is shown in “Figure 5” at different tem-
perature levels, using the measured weight of glycerol
as a conversion indicator for the yielded FAME. For the
samples investigated at room temperature (no process
heating), asymptotic FAME conversion value was not
reached within the time boundaries of this study. When
the in situ transesteriﬁcation process was carried out at
65˚C under the same reaction conditions, according to
“Table 4”, higher equilibrium conversion levels of
FAME of 43.1% and 76.22% were attained after reaction
time of 2 h and 4 h respectively.
Copyright © 2013 SciRes. JSBS
H. I. EL-SHIMI ET AL.
Table 3. Effect of catalyst concentration on biodiesel yield.
H2SO4 conc. H2SO4 vol. ml Yield %
30% 0.245 55.142
50% 0.41 77.23
100% 0.82 84.716
200% 1.64 84.716
Table 4. Effect of reaction time at different temperatures on
Time, hr 27˚C 40˚C 50˚C 65˚C
2 1.35 25.11 38.2 43.1
4 10.62 45.81 70.5 76.22
8 30.22 62.3 81.54 84.7
10 34.71 62.512 81.63 84.82
Figure 4. Effect of catalyst concentration on biodiesel yield
(at 65˚C for 8 hr using methanol-to-oil molar ratio of
Figure 5. Effect of reaction time at different temperatures
on FAME yield (using methanol-to-oil molar ratio of 3714:1,
100% wt. H2SO4 with constant stirring rate of 650 rpm).
The fact that the elevated temperatures improve the
initial miscibility of the reacting species, leading to a
signiﬁcant reduction in the reaction time, as observed in
Within the investigated experimental conditions, equi-
librium of FAME conversions was observed to reach
similar asymptotic values after a reaction time of 8 and
10 h for temperatures of 50 and 65˚C. Although faster
conversion rates could be observed by use of reaction
temperatures greater than the boiling point of the reacting
methanol (for example, 90˚C), the process heating and
pressure requirements may inhibit the use of such tem-
perature levels. The use of a reaction temperature of
65˚C may therefore prove more beneficial, if we consider
the total energy consumption and operation cost of the
whole biodiesel conversion system.
Temperature has detectable effect on the ultimate
conversion to ester. However, higher temperatures de-
crease the time required to reach maximum conversion.
The optimum temperature was 65˚C for 8 h. At lower
temperatures of 27˚C, the process was incomplete and no
FAMEs were observed.
3.5. Effect of Stirring
The stirring intensity appears to be of a particular impor-
tance for the alcoholysis process. Therefore, variations in
stirring intensity are expected to alter the kinetics of the
The effect of stirring on the in situ transesterification
process was performed as a potential process perform-
ance strategy. When the in situ transesteriﬁcation process
was conducted without stirring, no reaction would ob-
tained, and zero conversion of the microalgae oil content
to biodiesel is obtained, compared to that for the con-
tinuously stirred sample, “Table 5”. This indicates that
stirring is required to enhance the reaction progress, evi-
dently by aiding the initial miscibility of the reacting
species. However as illustrated in “Table 5”, after a re-
action time of 4 h under the same process conditions, the
samples stirred intermittently (1 h on and 1 h off) were
observed to achieve only 58.7% yield, and the FAME
yield achieved by the samples which were continuously
stirred was 76.22%, which prove the positive influence
of stirring during reaction.
3.6. Quality Assessment of Produced Biodiesel
Once biodiesel is obtained, a series of tests were con-
ducted to establish some properties of the produced bio-
diesel from microalgae. Viscosity, density, flash point,
cold flow properties and cetane number for produced
biodiesel in optimum conditions were measured by EN
methods. The obtained values compared to the EN 14214
standards have been shown in “Table 6”. Biodiesel
characteristics are strongly affected by the proportion of
long-chain and short-chain fatty acids and also the
presence of one or more double bonds . The viscosity
Copyright © 2013 SciRes. JSBS
H. I. EL-SHIMI ET AL. 231
Table 5. Effect of stirring on the in situ transesteriﬁcation of
microalgae lipids (at 65˚C for 4 hr with a H2SO4 concentra-
tion of 100% and methanol volume of 80 ml).
Stirring treatment Yield %
No stirring 0
Intermittently stirring (1 h off, 1 h on) 58.7
Continuously stirring 76.22
Table 6. Biodiesel properties: methods, limits and values.
Property Test - Method Lower limit Upper limitValue
EN ISO 3104 3.5 5.0 4.8
@ 15˚C) EN ISO 3675 860 900 886
Flash Point (˚C) ISO CD 3679e >101 - 172
(˚C) - - - 5
Pour Point (˚C) - - - −1
Cetane number EN ISO 5165 51 - 60.73
is one of the most important properties which affects the
fuel injection equipment and applied to determine the
conversion of microalgae oil to methyl-esters; since the
viscosity of produced biodiesel from microalgae was
determined to be 4.8 mm2/s and this value is much lower
than that of the crude microalgae oil which was 58 mm2/s.
Viscosity and density measurements of produced bio-
diesel are compliance with EN 14214 standards as shown
in “Table 6”, which confirm the biodiesel quality.
The flash point of produced microalgae biodiesel was
172˚C which exceeds the minimum flash point set by EN
14214 standards. This value is high as compared with
about 160˚C for jatropha biodiesel  and much higher
than that of 58˚C for petrol-diesel, which makes the
biodiesel, and its blends safer fuels to handle and store
near or with potential ignition sources.
The cloud and pour points of the produced biodiesel
are 5 and -1 respectively. These values are not better than
that given in literatures for sunflower biodiesel (2 and -3
respectively) and that for biodiesel from waste vegetable
oils (3 and -6 respectively); because the microalgae
methyl esters are mainly composed of saturated fatty
acids as illustrated in “Table 7”, and as stated by Alan
Scragg , the unsaturated fatty acids give better cold
flow properties than saturated fatty acids. Cetane number
of biodiesel is generally higher than conventional diesel
because it has longer fatty acids carbon chains and satu-
rated molecules. Microalgae biodiesel cetane number
was predicted from “Equation (4)” which conducted by
A.I. Bamgboye et al. .
Table 7. Fatty acids composition of microalgae biodiesel.
Fatty Acid % Composition (by wt.)
(X1) Lauric (C12:0) 0.7
(X2) Mystic (C14:0) 20.9
(X3) Palmitic (C16:0) 48.35
(X4) Stearic (C18:0) 2.02
(X5) Palmitoleic (C16:1) 2.66
(X6) Oleic (C18:1) 2.41
(X7) Linoleic (C18:2) 5.37
(X8) Linoleuic(C18:3) 7.84
where Xi, i= 1,2, …. 8, is the biodiesel fatty acids frac-
This formula gives approximate value for the biodiesel
cetane number as a function of biodiesel fatty acids
composition with accuracy of 90%. Fatty acids composi-
tion of the produced biodiesel is shown in “Table 7”.
Cetane number of microalgae biodiesel was calculated to
be 60.73, which is higher compared to 45.8 for rapeseed
biodiesel  and also better than 38 for jatropha bio-
diesel . The investigation of biodiesel cetane number
is of high importance; since inadequate cetane numbers
result in poor ignition quality, delay and excessive engine
This study investigated the effect of the most important
reaction variables on the conversion of Spirulina-Plat-
ensis microalgae oil to biodiesel using the acid-catalysed
in situ transesteriﬁcation process. Results show that
100% H2SO4 concentration (wt./wt oil) at 65˚C for 8 hr is
the optimum investigated conditions using 15 g of bio-
mass and 80 ml of the reacting methanol. The average
molecular weight of the Spirulina-Platensis oil was cal-
culated to be 845.19 g/mol., reduced to be 284 g/mol for
the produced FAME. Without stirring, no product will be
resulted. The properties of the produced fatty acid methyl
esters confirm the EN 14214 standards that make the
microalgae biodiesel a substitute fuel for petroleum-die-
The authors gratefully acknowledge financial support for
this research by FECU, Faculty of Engineering, Cairo
Copyright © 2013 SciRes. JSBS
H. I. EL-SHIMI ET AL.
 D. M. Huang, H. N. Zhou and L. Lin, “Biodiesel: An
Alternative to Conventional Fuel,” Energy Procedia, Vol.
16, Pt. C, 2012, pp. 1874-1885.
 A. Demirbas, “Biofuels Securing the Planet’s Future En-
ergy Needs,” Energy Conversion and Management, Vol.
50, No. 9, 2009, pp. 2239-2249.
 V. K. Wermelinger, S. Araujo, S. Hamacher and L. F.
Scavarda, “Economic Assessment of Biodiesel Produc-
tion from Waste Frying Oils,” Bioresource Technology,
Vol. 101, No. 12, 2010, pp. 4415-4422.
 L. Simasatitkul, R. Gani and A. Arpornwichanopa, “Op-
timal Design of Biodiesel Production Process from Waste
Cooking Palm Oil,” Procedia Engineering, Vol. 42, 2012,
 S. Nagarajan, S. K. Chou, S. Y. Cao, C. Wu and Z. Zhou,
“An Updated Comprehensive Techno-Economic Analysis
of Algae Biodiesel,” Bioresource Technology, 2012.
 C.-L. Cheng, P.-Y. Che, B.-Y. Chen, W.-J. Lee, C.-Y. Lin
and J.-S. Chang, “Biobutanol Production from Agricul-
tural Waste by an Acclimated Mixed Bacterial Micro-
ﬂora,” Applied Energy, Vol. 100, 2012, pp. 3-9.
 C. S. Jones and S. P. Mayﬁeldt, “Algae Biofuels: Versa-
tility for the Future of Bioenergy,” Current Opinion in
Biotechnology, Vol. 23, No. 3, 2012, pp. 346-351.
 K. Ramezani, S. Rowshanzamir and M. H. Eikani, “Cas-
tor oil Transesterification Reaction: A Kinetic Study and
Optimization of Parameters,” Energy, Vol. 35, No. 10,
2010, pp. 4142-4148. doi:10.1016/j.energy.2010.06.034
 B. B. Venu and V. G. Vaibhav, “Biodiesel Production
from Renewable Feed Stocks: Status and Opportunities,”
Renewable and Sustainable Energy Reviews, Vol. 16, No.
7, 2012, pp. 4763-4784. doi:10.1016/j.rser.2012.04.010
 F. Delrue, P.-A. Setier, C. Sahut, L. Cournac, A. Roubaud,
G. Peltier and A.-K. Froment, “An Economic, Sustain-
ability, and Energetic Model of Biodiesel Production
from Microalgae,” Bioresource Technology, Vol. 111,
2012, pp. 191-200. doi:10.1016/j.biortech.2012.02.020
 Y. Chisti, “Biodiesel from Microalgae,” Biotechnology
Advances, Vol. 25, No. 3, 2007, pp. 294-306.
 N. Nagle and P. Lemke, “Production of Methyl Ester
Fuels from Microalgae,” Applied Biochemistry and Bio-
technology, Vol. 24-25, No. 1, 1990, pp. 355-361.
 X. Miao and Q. Wu, “Biodiesel Production from Hetero-
trophic Microalgal Oil,” Bioresource Technology, Vol. 97,
No. 6, 2006, pp. 841-846.
 S. A. Abo El-Enin, N. K. Attia, N. N. El-Ibiari, G. I. El-
Diwani and K. M. El-Khatib, “In-Situ Transesteriﬁcation
of Rapeseed and Cost Indicators for Biodiesel Produc-
tion,” Renewable and Sustainable Energy Reviews, Vol.
18, 2013, pp. 471-477. doi:10.1016/j.rser.2012.10.033
 P. Chitra, P. Venkatachalam and A. Sampathrajan, “Op-
timisation of Experimental Conditions for Biodiesel Pro-
duction from Alkali-Catalysed Transesteriﬁcation of Jat-
ropha curcus Oil,” Energy Sustainable Development, Vol.
9, No. 3, 2005, pp. 13-18.
 Y. Zhang, M. A. Dub, D. D. McLean and M. Kates, “Bio-
diesel Production from Waste Cooking Oil: 2. Economic
Assessment and Sensitivity Analysis,” Bioresource Tech-
nology, Vol. 90, No. 3, 2003, pp. 229-240.
 S. Gryglewicz, “Rapeseed Oil Methyl Esters Preparation
Using Heterogeneous Catalysts,” Bioresource Technology,
Vol. 70, No. 3, 1999, pp. 249-253.
 S. Furuta, H. Matsuhashi and K. Arata, “Biodiesel Fuel
Production with Solid Super Acid Catalysis in ﬁxed Bed
Reactor under Atmospheric Pressure,” Catalyst Commu-
nication, Vol. 5, No. 12, 2004, pp. 721-723.
 H. Noureddini, X. Gao and R. S. Philkana, “Immobilized
Pseudomonas cepacia Lipase for Biodiesel Fuel Produc-
tion from Soybean Oil,” Bioresource Technology, Vol. 96,
No. 7, 2005, pp. 769-777.
 S. Hama, H. Yamaji, M. Kaieda, M. Oda, A. Kondo and
H. Fukuda, “Effect of Fatty Acid Membrane Composition
on Whole-Cell Biocatalysts for Biodiesel-Fuel Produc-
tion,” Biochemical Engineering Journal, Vol. 21, No. 2,
2004, pp. 155-160. doi:10.1016/j.bej.2004.05.009
 M. Oda, M. Kaieda, S. Hama, H. Yamaji, A. Kondo and
E. Izumoto, “Facilitator Effect of Immobilized Lipase-
Producing Rhizopus oryzae Cells on Acyl Migration in
Biodiesel-Fuel Production,” Biochemical Engineering
Journal, Vol. 23, No. 1, 2004, pp. 45-51.
 W. Du, Y. Xu, D. Liu and J. Zeng, “Comparative Study
on Lipase-Catalyzed Transformation of Soybean Oil for
Biodiesel Production with Different Acyl Acceptors,”
Journal of Molecular Catalysis B: Enzymatic, Vol. 30, No.
3-4, 2004, pp. 125-129.
 C. J. Shieh, H. F. Liao and C. C. Lee, “Optimization of
Lipase-Catalyzed Biodiesel by Response Surface Meth-
odology,” Bioresource Technology, Vol. 88, No. 2, 2003,
pp. 103-106. doi:10.1016/S0960-8524(02)00292-4
 E. A. Ehimen, Z. F. Sun and C. G. Carrington, “Variables
Affecting the in Situ Transesteriﬁcation of Microalgae
Lipids,” El-Sevier Fuel, Vol. 89, No. 3, 2010, pp. 677-
 S. Al-Zuhair, “Production of Biodiesel: Possibilities and
Challenges,” Biofuels Bioprod Bioreﬁning, Vol. 1, No. 1,
2007, pp. 57-66. doi:10.1002/bbb.2
 N. Kumar, “Biodiesel Production Technology and Feed-
stocks for India,” Expert Lecture at Workshop on Moving
Copyright © 2013 SciRes. JSBS
H. I. EL-SHIMI ET AL.
Copyright © 2013 SciRes. JSBS
toward Sustainable Energy Systems: Exploring Global
Pathways to a Common Destination Organized by Uni-
versity of Minnesota, Minneapolis, 2006.
 M. J. Haas, K. M. Scott, W. N. Marmer and T. A. Foglia,
“In Situ Alkaline Transesteriﬁcation: An Effective Method
for the Production of Fatty Acid Esters from Vegetable
Oils,” Journal of the American Oil Chemist’s Society, Vol.
81, No. 1, 2004, pp. 83-89.
 G. Hincapié, F. Mondragón and D. López, “Conventional
and in Situ Transesteriﬁcation of Castor Seed Oil for Bio-
diesel Production,” Fuel, Vol. 90, No. 4, 2011, pp. 1618-
 R. Halim, M. K. Danquah and P. A. Webley, “Extraction
of Oil from Microalgae for Biodiesel Production: A Re-
view,” Biotechnology Advances, Vol. 30, No. 3, 2012, pp.
 B. D. Wahlen, R. M. Willis and L. C. Seefeldt, “Biodiesel
Production by Simultaneous Extraction and Conversion
of Total Lipids from Microalgae, Cyanobacteria, and
Wild Mixed-Cultures,” Bioresource Technology, Vol.
102, No. 3, 2011, pp. 2724-2730.
 K. J. Harrington and C. D’Arcy-Evans, “A Comparison
of Conventional and in Situ Methods of Transesteri-
ﬁcation of Seed Oil from a Series of Sunﬂower Culti-
vars,” Journal of the American Oil Chemists’ Society, Vol.
62, No. 6, 1985, pp. 1009-1013.
 S. Siler-Marinkovic and A. Tomasevic, “Transesteriﬁca-
tion of Sunﬂower Oil in Situ,” Fuel, Vol. 77, No. 12,
1998, pp. 1389-1391.
 M. R. Andrade and J. A. V. Costa, “Mixotrophic Cultiva-
tion of Microalga Spirulina platensis Using Molasses as
Organic Substrate,” Biochemical Engineering Laboratory,
Department of Chemistry, Federal University Foundation
of Rio Grande (FURG), Rio Grande, 2006.
 L. H. Pelizer, E. D. G. Danesi, C. de O. Rangel, C. E. N.
Sassano, J. C. M. Carvalho, S. Sato and I. O. Moraes, “In-
fluence of Inoculum Age and Concentration in Spirulina
platensis Cultivation,” Departamento de Tecnologia Bio-
quımico-Farmaceutica, Faculdade de Ciencias Farmaceu-
ticas, Universidade de Sao Paulo, Sao Paulo, 2001.
 J. Sheng, R. Vannela and B. E. Rittmann, “Evalution of
Methods to Extract Andquantify Lipids from Synechocys-
tis PCC 6803,” Center for Environmental Biotechnology,
The Biodesign Institute, Arizona University, Tucson,
 Q. Hu, M. Sommerfeld, E. Jarvis, M. Ghiradi, M.
Posewitz, M. Siebert and A. Darzins, “Microalgal Triacy-
glycerols as Feedstock for Biofuel Production: Perspec-
tives and Advances,” The Plant Journal, Vol. 54, No. 4,
2008, pp. 621-639.
 A. A. Refaat, “Optimization of Biodiesel Production Us-
ing Different Techniques,” Master Thesis, Cairo Univer-
sity, Cairo, 2009.
 X. Meng, G. Chen and Y. Wang, “Biodiesel Production
from Waste Cooking Oil via Alkali Catalyst and Its En-
gine Test,” Fuel Processing Technology, Vol. 89, No. 9,
2008, pp. 851-857. doi:10.1016/j.fuproc.2008.02.006
 R. Zanzi, “Biodiesel from Microalgae,” B.Sc. Report
Submitted to Chemical Engineering and Technology De-
partment, Royal School of Technology, Stockholm, 2010.
 G. El-Diwani, N. K. Attia and S. I. Hawash, “Develop-
ment and Evaluation of Biodiesel Fuel and Byproducts
from Jatropha Oil,” International Journal of Environ-
mental Science and Technology, Vol. 6, No. 2, 2009, pp.
 A. Scragg, “Biofuels: Production, Application and De-
 A. I. Bamgboye and A. C. Hansen, “Prediction of Cetane
Number of Biodiesel Fuel from the Fatty Acid Methyl
Ester (FAME) Composition,” International Agrophysics,
Vol. 22, No. 1, 2008, pp. 21-29.
 J. M. Encinar, J. F. Gonzalez, A. Pardal and G. Martinez,
“Transesterification of Rapeseed Oil with Methanol in the
Presence of Various Co-Solvents,” Proceedings Venice
2010, Third International Symposium on Energy from
Biomass and WasteVenice, Italy, 8-11 November 2010.
 K. Sivaramakrishnan and P. Ravikumar, “Determination
of Cetane Number of Biodiesel and It’s Influence on Phy-
sical Properties,” ARPN Journal of Engineering and Ap-
plied Sciences, Vol. 7, No. 2, 2012, pp. 205-211.