Energy Conversion and Management xxx (2016) xxxxxx

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Energy Conversion and Management

journal homepage: www.elsevier.com/locate/enconman

Cultivation, extraction and optimization of biodiesel production from

potential microalgae Euglena sanguinea using eco-friendly natural
catalyst
Ajith J. Kings a, R. Edwin Raj a, L.R. Monisha Miriam b,, M. Adhi Visvanathan c
a
Department of Mechanical Engineering, St. Xaviers Catholic College of Engineering, Nagercoil 629003, India
b
Department of Biotechnology, Udaya School of Engineering, Vellamodi 629204, India
c
Pinnacle Biosciences, Algae Research and Development Center, South Thamaraikulam 629708, India

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

Article history: Algal biofuels have caught worldwide attention, due to the success of first and second generation biofuels
Available online xxxx on combing environmental concerns in spite of many challenges. Microalgae are the potential and sus-
tainable fuel source with carbon neutrality, where selection of promising strain is crucial for effective bio-
Keywords: fuel generation. Euglena sanguinea one such robust freshwater species with high lipid productivity was
Microalgae explored on laboratory scale as well as in mass scale in raceway pond. The extracted oils fatty acid value
Euglena sanguinea was only 3.8 mg KOH g
1 and had 93% triglycerides. Acid value was reduced to 0.3 mg KOH g
1 by
Optimization
esterification and the optimized parameters obtained were: methanol/oil ratio: 0.36, H2SO4: 10 vol.%,
Biocatalyst
Biodiesel
time: 42 min. In the subsequent transesterification process natural, recoverable and eco-friendly white
mussel shell was used. Maximum yield of 98% was obtained at 6 wt.% of calcinated CaO for the reaction
time of 80 min at 0.35 methanol/oil ratio. The GC-MS analysis of the algal biodiesel showed the presence
of saturated fatty acids: C16:0, C18:0, C22:0, C24:0 and monounsaturated fatty acids C18:1 at appropriate
level to provide better oxidation stability and combustion properties to use it for automotive application
without any major engine modifications.
2016 Elsevier Ltd. All rights reserved.

1. Introduction hope, but still needs comprehensive research to consolidate the

findings for commercialization [79].
The growing demand for fossil fuels due to the increase in vehi- Biodiesel is extracted by transesterification process in the pres-
cle population and industrialization raises the oil price because of ence of catalyst. Homogeneous catalysts such as NaOH, KOH are
its scarcity [1]. Moreover, the increasing environmental concerns generally used, in spite of issues in purifications, regenerability,
due to the greenhouse gas emission impacts the quality of human disposal and operational hazards due to its caustic and hygroscopic
life which needs to be addressed [2,3]. Exploring clean, renewable, nature [1012]. However, heterogeneous catalysts are non-
safe and sustainable substitute for petroleum based fuel is very corrosive in nature, reusable and are eco-friendly [13]. They do
decisive for meeting future energy needs [4]. not form soap through neutralization of fatty acid and saponifica-
Biodiesel produced from edible oil extracts such as rapeseed, tion of triglyceride [14]. The promising heterogeneous catalyst cal-
soybeans, palm oil, and sunflower oil faces criticism of food verses cium oxide (CaO) is used in this work for transesterification [15]. It
fuel dilemma [5]. To overcome this issue, non-edible feedstocks is non-toxic, low cost, highly active, long self-life and readily avail-
such as jatropha, mahuva, jojoba, tobacco seed, salmon oil, sea able [16]. It is synthesized from naturally available calcium
mango, waste cooking oil, restaurant greases, animal fats, pork resources such as egg shells [17,18], mollusk shells [19], chicken
lard, and beef tallow are used with its limitations [6]. However, bone [20], Oyster [21], and mud crab shell [22]. The raw source
microalgae is a potential biodiesel source due to its higher photo- undergoes the calcination process before it can be used as a cata-
synthetic efficiency leading to high lipid productivity with much lyst in the form of calcium oxide [23].
In this paper, aquatic microalgae Euglena sanguinea was culti-
vated in raceway ponds using organic compound medium with
Corresponding author. NPK fertilizer. An efficient low cost catalyst, calcinated from natu-
E-mail address: [email protected] (L.R.M. Miriam). ral white mussel shell at 1000 C was characterized and used in the

http://dx.doi.org/10.1016/j.enconman.2016.08.018
0196-8904/ 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Kings AJ et al. Cultivation, extraction and optimization of biodiesel production from potential microalgae Euglena san-
guinea using eco-friendly natural catalyst. Energy Convers Manage (2016), http://dx.doi.org/10.1016/j.enconman.2016.08.018
2 A.J. Kings et al. / Energy Conversion and Management xxx (2016) xxxxxx

conversion process. The process parameters were optimized to 0.5 kg of poultry litter was mixed in 5 l water and kept in a
minimize the acid value in the esterification process and then to closed container for fermentation at room temperature (30 C)
maximize the biodiesel yield in the transesterification process for 40 days. It was then filtered through 25 lm sieve and the fil-
using response surface methodology with lab scale experiments. trate was autoclaved at 120 C, 15 psi for 15 min and cooled. The
Finally, the FAME (fatty acid methyl esters) analysis was done composition of the filtrate is Nitrogen - 1.81%, Calcium - 1.1%, Iron
using GC-MS and the properties were compared with literature - 960 ppm, Manganese - 218 ppm, Zinc - 94 ppm, Copper - 27 ppm,
and standards. P2O5 - 1.47% and K2O - 0.98% separately.
100 g of seaweed (Gracilaria tenuistitata) was soaked in half litre
water and kept overnight for incubation. It was then heated to
2. Materials and methods 100 C for 6 h and the filtered using 25 lm mesh. The composition
of the extract is Carbohydrates - 5%, Protein - 0.5%, Lipids - 0.1%,
The process flow chart is shown in Fig. 1. Polyphenols - 0.25%, Nitrogen - 0.5%, Phosphorous - 0.68%,
Potassium - 2.0%, Calcium - 0.11%, Magnesium - 0.05%, Sodium -
2.1. Cultivation of microalgae 0.13%, Iron - 356 ppm, Zinc - 15.87 ppm, Copper - 17.62 ppm and
Manganese - 16.12 ppm along with traces of Cytokinin, Auxin,
The fresh water samples were collected from Vaadivilai (8110 N, Gibberelin [24].
77180 E), Kanyakumari District, Tamilnadu, India and filtered by 0.25 ml of the poultry litter filtrate solution was added to 0.5 ml
25 lm sieve. They were then enriched in Euglena medium having seaweed extract along with commercially available water soluble
a composition (g l
1): Sodium acetate 1, beef extract 1, bacto- fertilizer (all 19 NPK) of 0.5 ml to prepare the low cost organic
tryptone 2, yeast extract 2. Vitamins (mg l
1): vitamin compound medium. Culture was maintained in this medium at
B12 - 0.2; biotin - 0.1, thiamine (HCl) 100, niacin amide - 0.1. natural day/light setting with temperature variation between
The enriched samples were spread on a Euglena agar plate incu- 26 C and 32 C with a maximum day time light intensity of
bated at 30 C and were repeated until unialgal cultures of microal- 8000 lx. Then microalgae were cultivated in large scale with simi-
gae were obtained. The purified colonies were inoculated in 2 l lar conditions in raceway ponds (Fig. 2b) by frequently shaking it
Erlenmeyer flask with Euglena medium (pH 7) and incubated at gently using a paddle wheel rotated at 36 rpm. This species is pho-
30 C for a period of 15 days. Artificial photo period of 12:12 h totropic and heterotrophic in nature which can grow by using light
light/dark was provided using 3200 lx fluorescent light illumina- as the sole energy source or can utilize organic compound or both
tion. The cultures were shaken manually twice a day to avoid sed- for its energy source [25]. The purity of the culture was established
imentation of cells and microscopically observed for morphological by periodic microscopic observation and subculture. The log (max)
features such as size, shape and colour to establish culture purity. day was optimized as 12th day by observing its growth for 22 days.
So, the biomass were harvested on the log (max) day using 25 lm
sieves, dried in an oven and pulverized for oil extraction.
2.2. Mass propagation
2.3. Oil extraction and analysis
After initiating pure culture growth in the standard Euglena
medium, it was mass cultivated in a round cement tank with newly The oil was extracted from the powdered sample using a soxh-
derived low cost organic compound medium (poultry litter + sea- let extractor. The hexane: methanol (1:1) is used as the solvent
weed extract + all 19 NPK fertilizer) with culture volume of with microalgae powder in the ratio of 10:1 to achieve maximum
1000 l (Fig. 2a). lipid extraction [26]. The extraction was carried out for 6 h at a rate

Fig. 1. Step by step procedure of the conversion process of microalgae Euglena sanguinea to biodiesel.

Please cite this article in press as: Kings AJ et al. Cultivation, extraction and optimization of biodiesel production from potential microalgae Euglena san-
guinea using eco-friendly natural catalyst. Energy Convers Manage (2016), http://dx.doi.org/10.1016/j.enconman.2016.08.018
 A.J. Kings et al. / Energy Conversion and Management xxx (2016) xxxxxx 3

Fig. 2. Euglena cultivation in (a) round cement tanks (b) open raceway ponds.

of 8 cycles per hour at 70 C. Then the extract was filtered with to mass detector and capillary column, ELITE 1 (100 % Dimethyl
Whatman 40 filter paper and purified by simple distillation process poly siloxane) with nitrogen as a carrier gas with a flow rate of
to quantify algal oil. The free fatty acid (FFA) content, triglycerides, 1 ml min
1. The initial temperature of the oven was maintained
unsaponified matter (UNSAP) and the moisture content was ana- at 110 C for 2 min, then raised to 280 C at a heating rate of
lyzed using titrimetric procedure. 5 C/min and kept for 9 min. The FAME contents were detected
by comparing the peak areas with those that of the National Insti-
tute of Standards and Technology (NIST, 2005).
2.4. Biodiesel conversion
Biodiesel properties such as density, viscosity, flash point and
pour point were determined using standard equipments and com-
Two stage conversion processes was needed to convert the
pared to biodiesel standards. Other properties such as iodine value,
Euglena oil to biodiesel. In the esterification stage, the FFA content
cetane number, oxidation stability, phosphorous content, ash con-
was reduced within the limit and the process parameters such as
tent and cloud point were determined from the microalgae FAME
methanol/oil ratio, acid concentration, duration and stirring speed
profile [2729].
were designed to optimize them, while the temperature was main-
tained constant at 70 C. 100 ml three-neck round bottom flask
with rotomantle set up along with a reflux condenser, thermome- 3. Experimental design
ter and sampling port was used for lab scale experiments. The oil-
methanol mixture was taken along with the designed amount of Design of experiments (DoE) is a statistical tool employed to
H2SO4 and heated at 70 C with gentle stirring for a stipulated per- identify the major influencing factor (process parameter) and to
iod. It was then taken out and kept in a separating funnel for determine the optimum quantity to achieve the desired objective
15 min to separate the fatty acids and triglycerides from the [30]. Influencing process parameters and their ranges were identi-
acid-water solution. With these optimized conditions, sufficient fied from pilot experiments in corroboration with literature. The
quantity of oil was esterified using 1000 ml round bottom flask central composite design (CCD) is capable of handling nonlinear
for the transesterification process. relationships, when experiments are conducted in a randomized
Similar procedure was adopted here also with the same setup run order to protect against the effects of time-related variables
used for the esterification process. However, newly derived CaO and also to satisfy the statistical requirements. Response surface
biocatalyst was used instead of acid catalyst in the transesterifica- methodology was employed for statistical analysis of the experi-
tion process. The separation of biodiesel (methyl esters) from glyc- mental data using design experts 7.0 version software.
erine and CaO (heterogeneous catalyst) takes more than an hour.
The separated biodiesel was distilled at 180 C under a vacuum 3.1. Esterification process
pressure of
700 mmHg and the excess methanol was recovered
for reuse. In this work, white mussel shells were collected from A three level four factor central composite design was taken for
the Manakudi coastal area (850 N, 77280 E), Kanyakumari, India, this process with 30 sets of experiments including 6 replicates of
washed in fresh water and kept in an oven for 4 h at 110 C to the central points, 8 axial points and 16 factorial points. The levels
remove the moisture content. It was then powdered, sieved of the factors involved shown in Table 1. The RSM was used to opti-
through 200 lm mesh and calcinated. The calcinated compound mize the process parameters with an objective to minimize the
(CaO) was confirmed by powder X-ray diffraction (XRD) and FTIR acid value.
analysis.
3.2. Transesterification process
2.5. Characterization of biodiesel
A three level five factor central composite design was employed
FAME analysis was done by injecting 250 mg of algal biodiesel in this stage with 50 sets of experiments, consisting of 8 central
into gas chromatograph (GC Clarus 500 Perkin Elmer) connected points, 12 axial points and 30 factorial points. The RSM was

Please cite this article in press as: Kings AJ et al. Cultivation, extraction and optimization of biodiesel production from potential microalgae Euglena san-
guinea using eco-friendly natural catalyst. Energy Convers Manage (2016), http://dx.doi.org/10.1016/j.enconman.2016.08.018
4 A.J. Kings et al. / Energy Conversion and Management xxx (2016) xxxxxx

Table 1 bioreactor cultivation, even though the biomass productivity is

The range and levels of the esterification process parameters in biodiesel conversion. slightly low [34,35]. The microalga was cultivated in 10 raceway
Operating parameters Unit Ranges and levels pond of size 7.6  1.2  0.4 m with an average dried microalgae
Low (
1) Medium (0) High (+1) yield of 500 g day
1.
Methanol/oil (v/v) 0.25 0.35 0.45
H2SO4 (vol.%) 6 10 14 4.2. Analysis of E. sanguinea oil
Stirring speed rpm 350 625 900
Time min 30 40 50 The chemical composition of the extracted oil is as follows: Free
fatty acid 3.8 %, triglycerides 94.56 %, unsaponified matter 1.53 %
and moisture 0.11 %. Since the free fatty acid content is only
employed to optimize the parameters such as methanol to oil ratio, 3.8%, conversion of this algal lipid to biodiesel is quite easy which
catalyst concentration, temperature, reaction time and mixing requires simple esterification and transesterification process to
speed for maximizing the biodiesel yield. Process parameters and convert the oil into biodiesel.
their levels are tabulated in Table 2.
4.3. Characterization of biocatalyst
4. Results and discussion
In this study, the calcination time and temperature were opti-
4.1. Cultivation and extraction mized at 1000 C for 4 h. It is also reported that for egg shell
(1000 C and 2 h) [18], waste mud crab (Scylla serrata) shell (above
The collected sample observed through the microscope showed 700 C and 2 h) [22], natural white bivalve clam shell (900 C and
cylindrical algae with blunt ends. They are green in colour with red 4 h) [36], waste animal bone (1000 C and 4 h) [37], bovine bone
dots and are motile in nature, which confirms the microalgae spe- waste (above 950 C for 6 h) [38].
cies as Euglena sanguinea (Fig. 3). Moreover, the morphological fea-
tures are identical with the one reported in structure and 4.3.1. XRD analysis of mussel shell
reproduction of the algae by F.E. Fritsch. They are locally available The Fig. 4 shows the XRD pattern of the calcined white mussel
in plenty and are robust to climate variations. They belong to shells which are then compared with the standard peak records of
Euglenaceae family, which are known for rich oil content, are con- powder diffraction standards (JCPDS). Calcination converts calcium
sidered as a potential source for biodiesel extraction [3133]. carbonate to calcium oxide with the appearance of white colour.
Initially, the microalgae was cultivated in lab scale condition for The sharp major peaks at 2h of 18.023, 29.384, 34.084 and the
maintaining the pure culture and then cultivated in 1000 l round corresponding minor sharp peaks at 23.032, 28.668 31.418,
cement tanks and subsequently in raceway ponds with the condi- 35.952, 39.395, 43.144, 47.110, 47.396, 48.490, 50.790,
tions maintained in Sections 2.1 and 2.2 in large scale to harvest 54.345, 56.506, 57.351, 57.461, 59.370, 60.671, 61.340,
enough biomass. The harvested microalgae were kept in hot air 62.623, 64.262, 64.530, 65.563, 65.693, 70.215, 71.819,
oven at 60 C for 6 h. Enough quantity of algal oil was extracted 72.837, 77.135, 79.213 correspond to CaO and those at 13.112
by the procedure mentioned in Section 2.3., where an average of correspond to Ca(OH)2. The Ca(OH)2 phase appears due to the
25 ml of algal oil was obtained from 100 g of dried biomass. Careful exposure of CaO to air, which leads to the formation of Ca(OH)2.
control of pH and other physical parameters were done during cul-
tivation. Raceway pond cultivation is less expensive than photo- 4.3.2. FTIR analysis
FTIR analysis is used to analyze the chemical composition,
where Fig. 5 shows the one for the calcinated white mussel shell.
Table 2
The range and levels of the transesterification process parameters in biodiesel The major absorption well-defined peaks of the given sample are
conversion. at 3639 cm
1, 1421 cm
1 and 875 cm
1. A weak peak shown at
3746 cm
1 belongs to the NAH stretching, which is due to the
Operating parameters Unit Ranges and levels
presence of amino group. An explicit absorption peak shown at
Low (
1) Medium (0) High (+1)
3639 cm
1 is attributed to the OAH stretching due to the presence
Methanol/oil (v/v) 0.25 0.35 0.45 of hydroxyl group in Ca (OH) 2. This sharp peak confirms the pres-
Catalyst (wt.%) 3 6 9
ence of CaO and enables to compute the amount of CaO. The peak
Stirring speed rpm 400 600 800
Time min 50 80 110 at 1421 cm
1 indicates strong CAH bonding which shows the pres-
Temperature C 60 70 80 ence of CO3 in the sample. The band at 875 cm
1 corresponds to
asymmetric stretch due to OACAO bonds, resulting in the

Fig. 3. Microscopical observation of Euglena sanguinea at 400 (a) while moving (b) at the resting stage.

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 A.J. Kings et al. / Energy Conversion and Management xxx (2016) xxxxxx 5

Fig. 4. The XRD analysis results of white mussel shell after calcination.

major influence in reducing the acid value. However, the stirring

speed has relatively less influence with F-value of 45.
The perturbation plot (Fig. 6) relates the influence of all the
designed parameters to the acid value in the designed space by
varying a single parameter over its range while keeping the
remaining parameters constant at their midpoint value. The steep
slope of the curves with concave up shape indicates the signifi-
cance of the four identified process parameter and their range cov-
ering the optimized region. The interaction plot between
methanol/oil ratio with catalyst addition and reaction period
shows the interaction among the variables which necessitates
multi-variant approach for optimization (Fig. 7).
The three dimensional surface empirical model plots for acid
value which respect to the major two process parameters: metha-
nol/oil ratio and H2SO4 addition is shown in Fig. 8. The optimal
parameters were statistically predicted from the experimental
results with the criteria to reduce the acid value by using minimum
methanol and a catalyst (Table 4) which shows the minimum acid
Fig. 5. The FTIR analysis of the calcinated white mussel shell. value obtained when the catalyst concentration is slightly lower
than the predicted optimum value (10 vol.%). The acid value tends
to increase when the methanol/oil ratio is higher than the opti-
attachment of mono-dentate carbonate on the surface of CaO due
mum due to the back reaction of water in the presence of a cata-
to the limited contact of the catalyst with the air [39].
lyst. Similarly, extending the reaction period with vigorous
stirring facilitates free fatty acid formation [40,46].
4.4. Optimization of biodiesel conversion

4.4.1. Esterification 4.4.2. Transesterification

The extracted algal oil was heated at 110 C with smooth agita- The transesterification process converts the triglycerides of
tion for 1 h to remove the moisture content, since even the pres- algal oil into esters. Maximizing the ester yield by optimizing the
ence of 0.1% moisture will significantly decrease the conversion process parameters is the objective of this stage, where metha-
efficiency [40,41]. The objective of esterification process is to con- nol/oil ratio, catalyst concentration, reaction time, mixing speed
vert the free fatty acid (FFA) content into methyl esters because, and temperature are considered as the major influencing factors.
high amounts of FFA content leads to higher viscosity. The acid The parameter range and levels were finalized by pilot experi-
value should be less than 0.5 mg KOH g
1 in order to improve the ments. The designed sets of experiments were done and the results
biodiesel yield in transesterification process and also to prevent tabulated in Table 5.
soap formation [4245]. The Table 3 shows the designed process The perturbation plot (Fig. 9) relates the influence of all the
parameters for optimizing the same and to minimize the acid designed parameters to the biodiesel yield in the designed space
value. by varying a single parameter over its range while keeping the
The obtained acid values for the input parameters are analyzed remaining parameters constant at their midpoint value. The varia-
with design expert software to develop the empirical model. The tion in stirring speed (CAC arc) is not influencing much in the
model was tested for suitability and significance by Analysis of transesterification process; however, the other parameters have
Variance (ANNOVA) before navigating the designed space to draw much influence and are discussed below.
inferences. The F-value of the model was 361 and its p-value is less
than 0.0001 which implies the model significance. The F-value of 4.4.2.1. Effect of methanol/oil molar ratio. Methanol concentration is
the methanol/oil ratio, H2SO4 addition and the duration of the reac- one of the key factors in the conversion of triglycerides into methyl
tion (time) were 691, 531 and 486 respectively which signifies its esters. It is relatively cheaper and has high reactivity compared

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6 A.J. Kings et al. / Energy Conversion and Management xxx (2016) xxxxxx

Table 3
Designed process parameters and its corresponding acid values of the esterification process.

Sl. No Methanol/oil (v/v) H2SO4 (vol.%) Stirring speed (rpm) Time (min) Temperature (C) Acid value (mg KOH g
1)
1 0.25 6 350 50 70 1.83
2 0.35 10 625 60 70 1.43
3 0.25 6 900 50 70 1.62
4 0.25 6 900 30 70 1.92
5 0.25 14 350 30 70 1.44
6 0.45 14 350 50 70 0.85
7 0.55 10 625 40 70 0.59
8 0.35 10 625 40 70 0.34
9 0.45 14 900 50 70 0.65
10 0.45 14 350 30 70 1.3
11 0.45 6 900 30 70 1.63
12 0.45 6 900 50 70 0.9
13 0.15 10 625 40 70 1.59
14 0.25 6 350 30 70 1.92
15 0.35 10 625 40 70 0.37
16 0.45 6 350 50 70 0.92
17 0.35 10 625 40 70 0.39
18 0.35 10 75 40 70 1.24
19 0.35 10 625 40 70 0.29
20 0.25 14 350 50 70 1.39
21 0.35 18 625 40 70 0.81
22 0.25 14 900 30 70 1.24
23 0.35 10 625 40 70 0.33
24 0.35 10 625 40 70 0.35
25 0.35 10 1175 40 70 1.01
26 0.45 14 900 30 70 1.3
27 0.35 10 625 20 70 2.11
28 0.25 14 900 50 70 0.92
29 0.35 2 625 40 70 1.61
30 0.45 6 350 30 70 1.42

Fig. 6. The perturbation plot of the esterification process showing the influence of the process parameters on acid value.

with other alcohols, with a boiling point of 64.7 C, which makes it esterification process by simple distillation, which can be reused in
more suitable for biodiesel production [47,48]. The 3D surface plot the biodiesel conversion process or for other purposes.
and the corresponding contour diagram for the ester (biodiesel)
yield in relation with methanol/oil ratio and catalyst concentration 4.4.2.2. Effect of catalyst concentration. Alkaline catalyst is needed
is shown in Fig. 10a. Ester yield of around 98% was obtained even to facilitate the conversion of triglycerides into esters where natu-
at a methanol/oil ratio of 0.35, which was considered as the opti- ral CaO is used in the present study [50]. Insufficient amount of
mum minimum quantity. It can be noted that as the methanol/ catalyst, results in incomplete conversion within the stipulated
oil ratio increases the yield increases and gets saturated at the opti- time. However, the ester yield increases with CaO from 3 wt.%
mum limit [37]. The methanol increases the solubility, thus onwards and an optimum yield of around 98% was obtained when
encouraging the reaction with negligible mass transfer resistance the concentration was maintained between 6 wt.% and 8 wt.% by
[49]. Excess methanol was separated after esterification and trans- volume (Fig. 10a). Increasing the catalyst concentration beyond

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 A.J. Kings et al. / Energy Conversion and Management xxx (2016) xxxxxx 7

Fig. 7. The interaction plot among the esterification process parameter (a) the methanol/oil ratio and H2SO4 (b) the methanol/oil ratio and reaction period.

Fig. 8. The influence of the methanol/oil ratio and H2SO4 in the esterification process for minimizing the acid value (3D surface and contour plot).

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8 A.J. Kings et al. / Energy Conversion and Management xxx (2016) xxxxxx

Table 4 such as food, pharmaceutical, and cosmetics and in fuel cells for
The predicted optimized process parameters value for minimizing the acid value. power generation [53,54].
Methanol/oil H2SO4 Stirring speed Time Acid value
(v/v) (vol.%) (rpm) (rpm) (mg KOH g
1)
4.4.2.3. Effect of reaction temperature and time result. In general,
0.36 10 670 42 0.29
transesterification reaction is to be done at the lowest possible
temperature and time [55]. Temperature is a decisive factor in
the transesterification process, where low temperature increases
Table 5 the reaction time, which is not advisable for mass scale production
The designed process parameters of the transesterification process and the corre- [56]. Maintaining the process temperature just above its boiling
sponding ester yield. point (around 70 C) gives maximum yield (Fig. 10b). However, it
No. of Methanol/ Catalyst Speed Time Temperature Yield is important to maintain the rate of evaporation same as that of
run oil (v/v) (wt.%) (rpm) (min) (C) (%) reflux (condensation) to avoid methanol loss [56]. Moreover, the
1 0.35 6 600 80 45 27 refluxed methanol mixes more rapidly with oil enhancing the reac-
2 0.25 3 800 110 60 66.2 tion to yield maximum ester. Since transesterification is not a
3 0.45 3 800 50 60 56.8 reversible process, as the reaction time increases the yield of the
4 0.25 3 800 50 60 19.9
ester (biodiesel) increases. Fig. 8b also shows that the yield gets
5 0.25 3 800 50 80 37.5
6 0.25 9 400 110 60 73.3 maximum (98%) at around 80 min and further increases when
7 0.35 6 600 80 70 98.5 reaction time is redundant. Since the moisture content was
8 0.35 6 600 80 70 98.6 removed completely after the esterification process, increase of
9 0.25 3 400 110 80 73.2
reaction time has no effect on the ester yield, because soap forma-
10 0.45 9 800 50 80 78.5
11 0.25 9 400 50 60 36.7
tion does not take place in the absence of water in the transester-
12 0.35 6 600 80 70 98.4 ification process.
13 0.45 9 400 50 60 69.5
14 0.35 6 600 80 95 51.3
15 0.45 3 800 110 60 91.3 4.4.2.4. Effects of stirring speed. Stirring has a positive effect on the
16 0.45 9 400 110 80 95.8 yield of biodiesel even though it is not as pronounced as that of the
17 0.45 3 400 110 80 92.6
18 0.35 6 125 80 70 74.3
other factors. Proper mixing of oil with methanol is essential for
19 0.45 9 400 110 60 94.2 improving the biodiesel yield where the yield increases with the
20 0.45 3 400 50 80 66.5 speed of the stirrer up to 600 rpm. However, increasing the inten-
21 0.25 3 400 50 60 16.5 sity further affects the yield of biodiesel due to improper mixing
22 0.35 6 1075 80 70 77
[57,58].
23 0.35 6 600 80 70 98.5
24 0.45 9 400 50 80 79.6
25 0.35 6 600 80 70 98.6
26 0.25 9 400 50 80 54 4.5. Characterization of Euglena biodiesel
27 0.35 6 600 80 70 98.6
28 0.35 13 600 80 70 85.2 Basic characterization of biodiesel was done to ensure its
29 0.25 9 800 110 80 82.7 intended application in automobiles. The tested properties of
30 0.25 3 400 50 80 35.3
31 0.25 3 400 110 60 62.8
Euglena biodiesel were listed in Table 6 along with other reported
32 0.35 6 600 80 70 98.4 literature and standards. The density and viscosity were found to
33 0.25 3 800 110 80 75.5 be within the standard protocol of European and American norms
34 0.45 9 800 50 60 69.5 for biodiesel. Most of the properties such as cetane number, kine-
35 0.45 9 800 110 80 94.9
matic viscosity, oxidative stability and cold flow properties are
36 0.25 9 800 110 60 74.9
37 0.59 6 600 80 70 98.2 associated with the composition and structure of the fatty acid
38 0.25 9 400 110 80 82.2 profile of the biodiesel [5961].
39 0.11 6 600 80 70 37.7 GC-MS was done to identify and quantify the fatty acid com-
40 0.35 0 600 80 70 68.5 pounds present in the E. Sanguinea oil (Fig. 12). Most of the
41 0.45 3 800 110 80 93.3
42 0.35 6 600 150 70 98.4
microalgae have fatty acid compounds ranging from C11:0 to
43 0.25 9 800 50 80 54.5 C26:0 with the combination of polyunsaturated fatty acid (PUFA),
44 0.45 9 800 110 60 94.2 monounsaturated fatty acid (MUFA) and saturated fatty acid
45 0.45 3 400 50 60 54.9 (SFA) in a balanced quantity to provide enhanced biodiesel proper-
46 0.35 6 600 9 70 22.9
ties [62]. The GC analysis result of the extracted algae biodiesel
47 0.25 9 800 50 60 38.3
48 0.45 3 800 50 80 67.2 was C16:0, C18:0, C22:0, C24:0, which represents SFAs, C18:1 rep-
49 0.35 6 600 80 70 98.6 resents MUFAs and C18:2 belongs to PUFAs profile. The presence of
50 0.45 3 400 110 60 89.4 high amount of MUFA (52.24%) is advantageous because of its bet-
ter oxidation stability, stable cold flow phenomenon and enhanced
combustion characteristics [63]. It was also stated that a high pro-
portion of SFA increases the cloud point [64].
this range will not further increase the conversion efficiency to SFAs have full hydrogen atom that the carbon atoms can hold,
provide higher yield, because excess catalyst results in soap forma- and hence they do not have any double bonds between the carbon
tion [37,49,51]. atoms, whereas MUFAs have one double bond [65,66]. PUFAs have
The photograph shows the separated layer of biodiesel, glycer- more than one double bond. Saturated, long straight chain of ester
ine and the catalyst (Fig. 11). The CaO settles at the bottom of the provides a high cetane number regardless of branching [67]. The
separating funnel after the process, which can be recovered and high cetane number of microalgae based biodiesel is due to the
reused [52]. They can also be used along with the esterified acid presence of high amount of saturated and MUFAs, which reduces
catalyst to form gypsum, which is used as a fertilizer. The glycerine the ignition delay period thereby enhancing the combustion qual-
separated in the middle layer is recovered and used in industries ity [68,69].

Please cite this article in press as: Kings AJ et al. Cultivation, extraction and optimization of biodiesel production from potential microalgae Euglena san-
guinea using eco-friendly natural catalyst. Energy Convers Manage (2016), http://dx.doi.org/10.1016/j.enconman.2016.08.018
 A.J. Kings et al. / Energy Conversion and Management xxx (2016) xxxxxx 9

Fig. 9. The perturbation plot of the transesterification process showing the influence of process parameters on biodiesel yield.

Fig. 10. The influence of the transesterification process parameters on biodiesel yield (a) the methanol/oil ratio and catalyst concentration (b) the processing time and
reaction temperature.

Please cite this article in press as: Kings AJ et al. Cultivation, extraction and optimization of biodiesel production from potential microalgae Euglena san-
guinea using eco-friendly natural catalyst. Energy Convers Manage (2016), http://dx.doi.org/10.1016/j.enconman.2016.08.018
10 A.J. Kings et al. / Energy Conversion and Management xxx (2016) xxxxxx

4.6. Economical feasibility

Cost analysis with lab scale experiments is not comprehensive,

since prototype always cost much. However, the economical feasi-
bility of algal oil based biodiesel production was done with limited
conditions, costing $0.85/l. The cost contributions are approxi-
mately as follows: 70% for cultivation, harvesting, drying and pow-
dering; chemicals 10%, power 10% and the rest for miscellaneous.
Since low cost natural reusable heterogeneous catalyst was used,
the recovered products can be used for the production of gypsum,
methanol, etc. which will further reduce the cost of production.
The estimated cost of biodiesel will further reduce in large scale
production [73].

4.7. Conclusion

Universal energy crisis and environmental concerns have pro-

pelled the pursuit of sustainable fuel source with carbon neutrality.
In this intensive research hunt, microalgae proved its potential as a
sustainable biomass source for oil extraction. In this work, Euglena
sanguinea strain was isolated, cultivated and optimized on the lab
scale as well as in mass scale using raceway ponds. Biomass were
harvested on the log max day (12th) for oil extraction using soxhlet
method and then subjected to multi-variant approach to optimize
the biodiesel conversion process parameters. The acid value of raw
algal oil (3.8 mg KOH g
1) was reduced by esterification with
methanol induced by H2SO4 catalyst around to 0.3 mg KOH g
1
and further transesterified with eco-friendly biocatalyst, CaO to
methyl ester. The optimum methanol/oil ratio was obtained as
approximately 0.35 for both the stages and catalyst quantity as
10 vol.% and 6 wt.% for the first and second stage respectively.
The optimum reaction time for the first stage was 42 min and for
Fig. 11. Separation of crude biodiesel, glycerine and heterogeneous catalyst after the second stage, it was 80 min, where the biodiesel yield was
transesterification. max. at 98%. The stirring speed was found to be 670 rpm and

Table 6
The physio-chemical properties of Euglena biodiesel in comparison with other algae based biodiesel and standards.

Biodiesel properties Protocol Euglena Auxenochorella protothecoides Chlorella protothecoides Spirulina platensis
biodiesel [70] [71] [72]
EN ASTM
14214 D6751
Density at 15 C (kg m
3) 860900 875900 868 876.9 882 863.7
Viscosity at 40 C (mm2 s
1) 3.55.0 1.96 4.545 4.354 4.43 12.4
Flash point (C) Min. 120 Min. 93 172 160 189
Total sulphur (mg kg
1) Max. 10 Max. 15 <0.015 8.3  10
6 Nil
Cetane number Min. 51 Min. 47 65 52.6 70
Pour point (C)
15 to 10 13
9
Oxidation stability, 110 C (h) Min. 6 Min. 3 6.20 1.2 4.52
Acid value (mg KOH g
1) Max. 0.5 Max. 0.80 0.29 0.2 0.29 0.75
Iodine value Max. 120 118 112.2 102
Phosphorus content Max. 4 Max. 10 Nil
(mg kg
1)
Ash content (% mass) Max. Max 0.02 0.004 0.005 Nil
0.02
Cloud point (C)
3 to 12 15
Carbon residue (% mass) Max. 0.3 Max. 0.05 0.05 0.09 Nil

Fig. 12. The GC-MS analysis of the Euglena biodiesel.

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 A.J. Kings et al. / Energy Conversion and Management xxx (2016) xxxxxx 11

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