This document describes research on producing biodiesel from the marine microalga Chlorella salina using immobilized whole cell yeast. Some key points: - The yeast Rhodotorula mucilaginosa was immobilized on sugarcane bagasse to produce lipase enzymes for biodiesel production. - Oil was extracted from cultivated C. salina biomass and its molecular weight and fatty acid composition were analyzed. - The immobilized yeast cells were used as whole cell biocatalysts in a solvent-free system to convert the microalgal oil to biodiesel via interesterification, optimizing various reaction parameters like biocatalyst loading, temperature, and water content

1. Journal of Environmental Chemical Engineering 2 (2014) 1294–1300
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Journal of Environmental Chemical Engineering
journal homepage: www.elsevier.com/locate/jece
Biodiesel production from marine microalga Chlorella salina using
whole cell yeast immobilized on sugarcane bagasse
Duraiarasan Surendhiran*
, Mani Vijay, Abdul Razack Sirajunnisa
Bioelectrochemical Laboratory, Department of Chemical Engineering, Annamalai University, Annamalainagar, Tamil Nadu 608002, India
a r t i c l e i n f o
Article history:
Received 20 January 2014
Accepted 7 May 2014
Keywords:
Chlorella salina
Biodiesel
Methyl acetate
Whole cell biocatalyst
Interesterification
a b s t r a c t
Nowadays microalgae have become a potential source for production of biodiesel due to its fast growth
rate, high lipid content and incapable of affecting the food chain. In this study immobilized whole cell yeast
Rhodotorula mucilaginosa MTCC8737 was employed for conversion of marine microalga Chlorella salina oil
into biodiesel by non-alcoholic route in a solvent-free system. Various parameters were evaluated to enhance
the biodiesel yield with methyl acetate as an acyl acceptor. The maximum biodiesel yield was obtained at
85.29% with the optimum conditions of 1.5 g whole cell biocatalyst, 1:12 methyl acetate to oil ratio, 10%
water content (w/w), temperature of 40 ◦
C, 60 h of reaction time and agitation at 250 rpm. The stability of
immobilized whole cell biocatalyst was studied with 10 cycles of repeated usage and it was shown that there
was no significant loss of lipase activity in the presence of methyl acetate. The fatty acid composition was
analyzed by gas chromatography, which resulted that palmitic (C16:0) and oleic acid (C18:1) are predominant
in C. salina biodiesel. This study proved that the use of whole cell yeast immobilized on sugarcane bagasse
is cost-effective, ecofriendly and an alternative method for enzymatic biodiesel production on a commercial
scale.
c 2014 Elsevier Ltd. All rights reserved.
Introduction
Currently there is a worldwide interest in finding out new alter-
native fuels against fossil fuels because of diminishing and over con-
sumption of hydrocarbons which, result in the accumulation of green
house gases in atmosphere that ultimately leads to global warming
[1,2]. Biodiesel (monoalkyl esters of long chain fatty acids) is a po-
tential renewable biofuel and it is biodegradable, non-toxic, has no
net carbon dioxide and is free of sulfur [3–6]. Generally, biodiesel is
produced from food materials and oil crops using conventional meth-
ods [7]; however these sources cannot realistically replace the wide
use of diesel fuel due to increasing demand. Also the over population
worldwide has lead to serious land shortage and raised the issue of
food security [8]. Microalgae have become a recent attraction because
of high oil content, and can be grown in wastewater as they do not
compete with food crops for arable land and water and give 20 times
more biomass productivity rate than the terrestrial crops [9–14]. Mi-
croalgae are photosynthetic microorganisms that utilize light, water
and CO2 and accumulate intracellular lipids as storage materials [15].
Currently biodiesel is being produced employing conventional
methods such as acid and alkali transesterification that results in the
conversion of triglycerides into fatty acid methyl esters in a shorter
period [16,17]. Major drawbacks of conventional methods include
high energy input, elimination of salt, difficulty in recycling glycerol,
* Corresponding author.
E-mail address: suren micro@yahoo.co.in (D. Surendhiran).
soap formation and requiring wastewater treatment [18–25]. To over-
come these problems, enzymatic production of biodiesel has recently
become an alternative for biodiesel production because the byprod-
uct glycerol can be easily recovered, salt and catalyst can be avoided,
wastewater treatment is not required, high production yield could
be attained under milder conditions and it is an ecofriendly process
[26–28]. One such enzymes used in biodiesel production are lipases.
Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are produced by mi-
croorganisms, plants and animals, but for the large scale production
microorganisms are more suitable [29].
However, the enzymatic production of biodiesel is not yet com-
mercialized due to high cost involvement in the isolation, purification,
and immobilization on a carrier as well as to low stability of lipase in
methanol [30–33]. To overcome these problems, we have focused on
whole cell biocatalyst for biodiesel production, i.e. microorganisms,
which contain lipase intracellularly or in their cell wall. The advan-
tages of whole cell biocatalyst include inexpensive process, recycla-
bility of biocatalyst, no purification process, stability to methanol and
low production cost [15,18,25]. Nowadays, the enzymatic synthesis
of biodiesel in solvent free system is focused globally, because such
systems are advantageous over solvent aided transesterification by
avoiding separation, toxicity, flammability and high cost of organic
solvents [20,33].
In this work, biodiesel had been produced by interesterification of
oil from microalga, Chlorella salina. Biocatalysis of the reaction was
performed by whole cells of Rhodotorula mucilaginosa producing li-
pase which was immobilized on an agro waste, sugarcane bagasse. As
2213-3437/$ - see front matter c 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jece.2014.05.004

2. D. Surendhiran et al. / Journal of Environmental Chemical Engineering 2 (2014) 1294–1300 1295
India is one of the largest sugarcane producing countries, sugarcane
bagasse is one of the abundantly available raw material for most of the
biotechnological productions and also, it is cheap and biodegradable.
Hence, sugarcane bagasse had been used as the supporting material
for this study. From an extensive literature survey it was found that
there was no report on whole cell immobilized on sugarcane bagasse
for biodiesel production, this being the first report.
Materials and methods
Culture condition
C. salina was obtained from CMFRI, Tuticorin, Tamilnadu, India
and cultivated in 200 l photobioreactor (PBR) using sterile Walne’s
medium. The filtered sterilized seawater was enriched with re-
quired quantity of Walne’s medium containing (g/l): NaNO3, 100;
NaH2PO4·2H2O, 20.0; Na2EDTA, 4.0; H3BO3, 33.6; MnCl2·4H2O, 0.36;
FeCl3·6H2O, 13.0; vitamin B12, 0.001 and vitamin B1, 0.02. The trace
metal solution contained (g/l): ZnSO4·7H2O, 4.4; CoCl2·6H2O, 2.0;
(NH4)6Mo7O24·H2O, 0.9; and CuSO4·5H2O, 2.0. The medium was ad-
justed to pH 8 and autoclaved at 121 ◦C for 20 min. The filter sterilized
vitamins were added after cooling [34,35]. Mixing was provided by
sparging air from the bottom of the PBR and lighting was supplied by
cool-white fluorescent light with an intensity of 5000 lx under 12/12
light/dark cycle for 15 days.
Microorganism and culture condition
The lipase producing yeast R. mucilaginosa MTCC8737 was ob-
tained from IMTECH, Chandigarh, India. The culture was subcultured
using YPD growth medium containing malt extract 3 g, yeast extract
3 g, peptone 5 g, glucose 10 g/l with 1% olive oil. The pH was main-
tained at 6.2 and incubated at 30 ◦C for 48 h at 200 rpm. The yeast cells
were harvested by centrifugation and the pellets were washed with
50 mM Tris–HCl buffer. The pellets and supernatant were subjected
to lipase and protein assays.
Preparation of whole cell biocatalyst using sugarcane bagasse
Sugarcane bagasse was collected from local juice shop near An-
namalai University campus and cut into 5 mm size. The sugarcane
bagasse chips were washed with distilled water and dried at 100 ◦C
up to constant weight. The dried chips were subjected to alkaline
pretreatment to increasing affinity between yeast cells and bagasse.
20 g of dried bagasse was sterilized by autoclave and mixed with
0.5 M NaOH solution in Erlenmeyer flask, then incubated in a rotary
shaker at 120 rpm for 24 h [36]. Finally it was washed again with
sterilized water and dried under same conditions. Sugarcane bagasse
chips were suspended in 200 ml of YPD medium containing yeast cells
R. mucilaginosa MTCC8737 for whole cell immobilization. The mixture
was incubated for 16 h for growth and adsorption [37]. Then the im-
mobilized biocatalyst was removed from culture medium, stained
using crystal violet, observed under light microscope and used for the
biodiesel production.
Harvesting of microalgal biomass and oil extraction
When the culture reached stationary phase, the biomass was har-
vested using marine Bacillus subtilis (MTCC 10,619) to get thick mi-
croalgal paste as reported in our previous work [38]. Then the microal-
gal paste was rinsed with distilled water to remove residual salts and
then dried in hot air oven at 60 ◦C for 8 h. Dried biomass was subjected
to oil extraction by Bligh and Dyer [39] with slight modification. In
brief, the biomass suspension was mixed with chloroform: methanol
(1:2) ratio, vortex it for few minutes and incubated on ice for 10 min.
Then, chloroform was added followed by addition of 1 M HCl and
again vortexed it for few minutes. Finally the whole suspension was
centrifuged at maximum speed for 2 min. Bottom layer containing
lipid was transferred into fresh previously weighed beaker. Chloro-
form was added to reextract the lipid from the aqueous sample. The
solvent system was evaporated using rotary evaporator at 30 ◦C.
Lipase assay and protein determination
Lipase activity was determined for culture supernatant according
to Burkert et al. [40] and Padilha et al. [41]. The olive oil emulsion
was prepared by mixing 25 ml of olive oil and 75 ml of 7% Arabic gum
solution in a homogenizer for 5 min at 500 rpm. The reaction mixture
containing 5 ml of emulsion, 2 ml of 10 mM phosphate buffer (pH 7.0)
and 1 ml of the culture supernatant was incubated at 37 ◦C for 30 min
in orbital shaker. The reaction was stopped by addition of 15 ml of
acetone–ethanol (1:1, v/v), and the liberated fatty acids were titrated
with 0.05 N NaOH. One unit of lipase activity was defined as the
amount of enzyme, which liberated 1 μmol of fatty acid per minute.
The protein content in the crude enzyme was determined by Lowry
et al. [42] with BSA as a standard.
Determination of molecular weight of microalgal oil
According to Sathasivam and Manickam [43] the saponification
and acid value of microalgal oil were determined. The molecular
weight of the oil was calculated as [44]:
M =
168,300
SV − AV
where M is the molecular weight of the oil and SV the saponification
value and AV is the acid value.
Optimization of enzyme interesterification process by solvent-free
system
The enzymatic transesterification reaction was carried out in 15 ml
screw cap glass vial. No solvent was added in this reaction. The re-
action mixture consisted of 3 g of microalgal oil, 1 g of immobilized
whole cell biocatalyst and methyl acetate. The oil to acyl acceptor
(methyl acetate) was optimized ranging from 1:2, 1:4, 1:6, 1:8, 1:10,
1:12 and 1:14. The effect of temperature was studied at various in-
tervals of 25, 30, 35, 40 and 45 ◦C. In order to investigate the effect of
water enzymatic transesterification was carried out by adding small
amount of water at the concentration of 0, 2, 4, 6, 8, 10 and 12 wt% of
the total amount of reaction mixture. The transesterification reaction
was allowed for 48 h at constant speed of 200 rpm. The biodiesel yield
was calculated according to Umdu et al. [45]:
Biodiesel yield (wt%) =
(Amount of biodiesel (g) in upper mixture)
(Amount of microalgal oil (g))
× 100
GC analysis of fatty acid methyl esters
Fatty acid methyl ester composition of biodiesel produced from
C. salina oil was analyzed by gas chromatography–mass spectrom-
etry (GC-MS-QP 2010, Shimadzu) equipped with VF-5 MS capillary
column (30 mm length, 0.25 mm diameter and 0.25 μm film thick-
ness). The column temperature of each run was started at 70 ◦C for
3 min, then raised to 300 ◦C and maintained at 300 ◦C for 9 min. GC
conditions were: column oven temperature − 70 ◦C, injector tempera-
ture − 240 ◦C, injection mode split, split ratio – 10, flow control mode
– linear velocity, column flow – 1.51 ml/min, carrier gas – helium
(99.9995% purity) and injection volume – 1 μl. MS conditions were:
ion source temperature − 200 ◦C, interface temperature − 240 ◦C,
scan range – 40–1000 m/z, solvent cut time – 5 min, MS start time –
5 min, end time – 35 min and ionization – EI (−70 eV) and scan speed
– 2000.

3. 1296 D. Surendhiran et al. / Journal of Environmental Chemical Engineering 2 (2014) 1294–1300
Fig. 1. Immobilization of yeast budding cells on biomass support-sugarcane bagasse.
Results and discussion
Quantification and characterization of microalgal oil
The oil content of C. salina was calculated according to Suganya
and Renganathan [46] and the yield of oil extracted was found to be
28.26% (w/w). The lipid concentration was defined as dry weight ratio
of extracted lipids to biomass. The molecular weight of C. salina oil
was 849.88 (g/mol), which was calculated using acid value (0.42 mg
KOH g−1) and saponification value (198.45 mg KOH g−1). The iodine
value was found to be 65 (mg/g).
Quantification of lipase assay
1% olive oil was used for enhancing lipase production. The activity
of lipase from the yeast R. mucilaginosa MTCC8737 was found to be
1.26 U ml−1.
Effect of biocatalyst loading
The cost of the enzyme involved in biodiesel generation affects the
overall economy of the production. Hence quantity of enzyme used
for the process should be mitigated [47]. In this study, effect of catalyst
was investigated for the interesterification reaction, and whole yeast
cells were used as biocatalyst adsorbed on sugar cane bagasse (Fig.
1). The surface area of sugar cane bagasse was found to be 1785 m2/g
which was analyzed using Mastersizer 2000, Malvern Instruments,
UK. Effect of whole cell biocatalyst loading was studied to perform in-
teresterification in the range of 0.5 and 2.5 g. Fig. 2 showed that 1.5 g
of biocatalyst load yielded maximum biodiesel. The study revealed
that higher cell concentration produced more biodiesel but above the
optimal level of 1.5 g, the yield decreased. The result was in com-
plete agreement with Arumugam and Ponnusami [48]. This might be
due to obstruction of mass transfer by larger particles. Moreover, the
superfluous enzyme would unite and retard lipase activity [49].
Effect of oil and methyl acetate molar ratio
Oil to methyl acetate ratio is one of the key factors for biodiesel
production. The study showed that molar ratio 1:12 imparted largest
biodiesel yield of 56.2% at 48 h in the absence of solvents (Fig. 3).
This report was consistent with Ognjanovic et al. [20]. As the mo-
lar ratio was raised to 1:14, a decline in production was observed.
The poor yield could be due to the presence of excess amount of
methyl acetate that would dilute the oil [50]. Ratios lesser to the op-
timal value also exhibited insufficient yield. The conventional short
chains namely methanol or ethanol inactivated lipase above molar
Fig. 2. Effect of whole cell biocatalyst dosage on biodiesel yield (%). Reaction parame-
ters: 1:4 M ratio of methyl acetate to oil, 30 ◦
C, 200 rpm and 48 h.
Fig. 3. Effect of molar ratio of methyl acetate to microalgal oil on biodiesel yield (%).
Reaction parameters: 3 g whole cell biocatalyst, 30 ◦
C, 200 rpm and 48 h.
ratio of 1:3. Similarly Shimada et al. [51] had reported that immobi-
lized lipase Novozym 435 from Candidaantarctica was inactivated at
the molar ratio of 1:5 of plant oil and methanol. In addition, during
methanolic transesterification, the prime byproduct glycerol, which
is hydrophilic and immiscible in oil, results in low reactivity of the cat-
alyst due to mass transfer resistance. In contrary, this study involved
methyl acetate, which produced triacylglycerol instead of glycerol,
which do not inactivate lipase [53].
Effect of temperature
Temperature is specific to each enzyme and its functions. It is to
be noted that biodiesel yield is proportional to the temperature in-
volved; at a low temperature, a slow activity is evinced, and as the
temperature increased the reaction rate also increased with an effec-
tive increase in biodiesel production [54]. The higher the temperature
and lipase collision with substrate molecules, the higher would be the
reaction rate [55]. Most of the reaction involving enzymes would fol-
low Arrhenius equation at low temperature, but at high temperature
yield is enhanced. Moreover, in certain cases, thermal denaturation
of the enzyme might occur with elevation of temperature, thus trans-
formation of oil to FAME gets negatively affected [54–57]. In order to
study the effect of temperature an enzymatic biodiesel process, the
range studied was between 20–45 ◦C with an interval of 5 ◦C. The re-
sults showed that 40 ◦C gave the highest yield of 68.81% (Fig.4). When
the temperature exceeded 40 ◦C, the yeast enzyme was apparently
inactivated, thus yielding a low biodiesel quantity. However, most
of the enzymatic reactions do not require higher temperature [17].
Since higher temperature need not be implemented, there would be
no high expenditure of energy, which is an advantage of the current

4. D. Surendhiran et al. / Journal of Environmental Chemical Engineering 2 (2014) 1294–1300 1297
Fig. 4. Effect of temperature on biodiesel yield (%). Reaction parameters: 3 g whole
cell biocatalyst, methyl acetate to oil ratio 1:12, 200 rpm and 48 h.
Fig. 5. Effect of water on biodiesel yield (%). Reaction parameters: 3 g whole cell
biocatalyst, methyl acetate to oil ratio 1:12, 40 ◦
C, 200 rpm and 48 h.
findings.
Effect of water
Water is one of the essential factors in biotransformation of oil to
biodiesel. Existence of water in the reaction mixture would avoid
lipase deactivation [57]. Lipase hydrolyses triglycerides in the in-
terfacial area. Lipase efficiently catalyses hydrolysis in the aqueous
medium, perhaps the reaction gets induce in excess amount of water.
The enzyme usually is active at the interfacial area between aqueous
and organic phase; the activity is depended upon it. Increase in water
content leads to more oil water droplets within an oil–water system,
eventually resulting in higher interfacial area. Optimum water con-
tent reduces hydrolytic reaction and elevates enzyme reaction during
transesterification [48,58]. In the present study, effect of water was
studied by carrying out reactions at varying concentrations of 0–12%.
Maximum yield was achieved with 10% of water (Fig. 5). Moreover,
there was no decrease in methyl esters until 10% was reached, since
formation of triacylglycerol occurred which did not disturb the ac-
tivity of lipase. Lee and Yan [4] had similarly reported that presence
of water over 7% of the total reaction mixture deteriorated biodiesel
formation. When water content reached beyond 10% yield decreased.
This decline could be due to hydrolysis of FAME as given in previ-
ous study [57]. In additional, more quantity of water might affect
mass transfer of oil and methyl esters through aqueous phase, hence
lowering the production [48].
Fig. 6. Effect of reaction time on biodiesel yield (%). Reaction parameters: 3 g whole
cell biocatalyst, methyl acetate to oil ratio 1:12, 10% water (w/w) 40 ◦
C, 200 rpm and
48 h.
Fig. 7. Effect of agitation on biodiesel yield (%). Reaction parameters: 3 g whole cell
biocatalyst, methyl acetate to oil ratio 1:12, 10% water (w/w) 40 ◦
C and 60 h.
Effect of reaction time on biodiesel yield
Effect of reaction time was studied in the range of 12 and 72 h with
an interval of 12 h. The time taken for maximum biocatalysis of oil
to biodiesel was observed at 60 h. As the reaction time was increased
beyond the optimal range, a decrease in biodiesel production was
obtained (Fig. 6). This might attribute towards hydrolysis of biodiesel,
which would be triggered by the excess water [4].
Effect of agitation on biodiesel yield
Agitation is one of the most important parameters in interesteri-
fication process. During immobilization, reactants diffuse to external
environment from the bulk liquid ultimately into the internal pores
of the enzyme [52]. Effect of mixing on biodiesel production was con-
ducted between 100 and 400 rpm with an interval of 100 rpm. Fig.7
shows the methyl esters production rate to their respective speed of
agitation. The maximum yield of biodiesel was found to be 300 rpm,
which indicated that agitation enhances the rate of reaction. Gener-
ally, agitation mitigates mass transfer resistance between oil and acyl
acceptor and immobilized lipase at the interface of catalysis, thus en-
hancing the rate of reaction. When agitation speed crossed 300 rpm,
yield decreased. This might be because of the high mechanical shear
which results in biocatalyst distortion leading to inactivation of lipase
[4,50,52].
Reusability of whole cell biocatalyst
Industrial operation of enzymatic biodiesel production is very dif-
ficult due to high cost of lipase [59]. Reusability of the biocatalyst is

5. 1298 D. Surendhiran et al. / Journal of Environmental Chemical Engineering 2 (2014) 1294–1300
Table 1
Fatty acid composition of C. salina FAME.
Lipid number Common name Systematic name Molecular structure Fatty acid (%)
C14:0 Myristic acid Tetradecanoic acid C14H28O2 3.00
C16:0 Palmitic acid Hexadecanoic acid C16H32O2 23.85
C16:1 Palmitoleic acid 9-Hexadecanoic acid C16H30O2 11.18
C18:0 Stearic acid Octadecanoic acid C18H36O2 9.55
C18:1 Oleic acid 9-Octadecenoic acid C18H34O2 40.77
C18:2 Linoleic acid 9,12-Octadecadienoic acid C18H32O2 11.65
Table 2
Comparison of physio-chemical properties of biodiesel from C. salina with petrodiesel and jatropha biodiesel.
Properties Diesel fuel Biodiesel from jatropha Biodiesel from C. salina
Density (g/ml) 0.841 0.865 0.864
Kinematic viscosity (@ 40 ◦
C) 1.9–4.5 5.2 5.6
Flash point (◦
C) 50–80 175 178
Fire point (◦
C) 78 136 149
Pour point (◦
C) −6 −2 −4
Fig. 8. Reusability and stability of whole cell biocatalyst on biodiesel yield (%). Reaction
parameters: 3 g whole cell biocatalyst, methyl acetate to oil ratio 1:12, 10% water (w/
w) 40 ◦
C, 250 rpm and 60 h.
one of the most advantageous phenomena of immobilized enzyme.
This is another major parameter influencing the overall production
economy. Usually, stable and recyclable biocatalyst retards the cost of
generation of biodiesel from oil [48,60,61]. This factor decides the pos-
sibility of large scale production of biodiesel utilizing enzymes [28].
In this section, stability and reusability of immobilized biocatalyst,
whole cells of R. mucilaginosa was investigated. The study revealed
that there was no significant loss in activity of enzyme even after
utilizing after 10 cycles (Fig. 8). This study was in contrast to that
of Srimhan et al. [3], which reported that yield, was decreased from
83.29 to 59.31 in the second cycle of bioconversion in the presence of
methanol. But according to Du et al. [62], whose results were in agree-
ment to the present investigation showed that there was no loss of
biocatalyst even after 100 cycles of repeated usage in the presence
of methyl acetate. Methanol or ethanol, when used as acyl acceptor,
produces glycerol as the byproduct, whose removal is intensive, and
costly consuming and inactivates lipase. Thus, the present study in-
dicated that immobilized lipase could be used for many cycles with
methyl acetate as acyl acceptor that finally reduces the cost of overall
bioconversion process.
Fatty acid composition of C. salina FAME
Table 1 shows the six fatty acids present in the C. salina biodiesel.
From the retention time obtained by GC-MS, peak values were ana-
lyzed and observed as myristic acid (C14:0), palmitic acid (C16:0),
palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1)
Fig. 9. Gas chromatogram of FAME obtained from C. salina.
and linoleic acid (C18:2), which were commonly found in C. salina
biodiesel (Fig. 9). Moreover, palmitic acid and oleic acid are predom-
inant in the C. salina biodiesel content synthesized by enzymatic in-
teresterification. For C. salina, the oleic acid content was slightly in-
creased from 40.77% to 48.14%. Feng et al. [63] reported that high con-
tent of oleic acid is relatively suitable for biodiesel. Many researchers
reported that the biodiesel cannot be stored for a long period be-
cause of its oxidation sensitive in nature. But the high levels of oleic
acid content make the biodiesel highly oxidation stable [64]. Since C.
salina contains more amount of oleic acid than the other fatty acids,
it could be a promising source for biodiesel production and resistant
to oxidation.
Properties of biodiesel from C. salina
The physio-chemical properties of C. salina biodiesel synthesized
through interesterification are listed in Table 2. The results were com-
pared with that of diesel fuel and biodiesel from jatropha oil as stated
by ASTM standard D6751. The final results revealed that no substan-
tial variations were observed between biodiesel properties of C. salina
and jatropha oil.
Conclusion
Biodiesel production from marine microalgae C. salina was in-
vestigated using whole cell yeast as biocatalyst. The yeast cell was
successfully immobilized on an agro waste sugarcane bagasse and
the maximum yield was found to be 85.29% with influential parame-
ters such as biocatalyst loading, molar ratio of oil to methyl acetate,
temperature, water content, reaction time and agitation. The current
study showed that the properties of obtained microalgal biodiesel
fulfilled the standards of ASTM (D6751). The whole cell biocatalyst

6. D. Surendhiran et al. / Journal of Environmental Chemical Engineering 2 (2014) 1294–1300 1299
R. mucilaginosa MTCC8737 showed good stability in repeated cycles
without significant loss of its activity and proved that this method
could be economically feasible for reducing the cost of enzymatic
production of biodiesel.
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