Journal of Environmental Chemical Engineering 2 (2014) 1294–1300

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Contents lists available at ScienceDirect

Journal of Environmental Chemical Engineering

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j e c e

iodiesel production from marine microalga Chlorella salina using

hole cell yeast immobilized on sugarcane bagasse

uraiarasan Surendhiran

* , Mani Vijay, Abdul Razack Sirajunnisa

ioelectrochemical Laboratory, Department of Chemical Engineering, Annamalai University, Annamalainagar, Tamil Nadu 608002, India

r t i c l e i n f o

rticle history:

eceived 20 January 2014

ccepted 7 May 2014

eywords:

hlorella salina

iodiesel

ethyl acetate

hole cell biocatalyst

nteresterification

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.

ntroduction

Currently there is a worldwide interest in finding out new alter-

ative fuels against fossil fuels because of diminishing and over con-

umption of hydrocarbons which, result in the accumulation of green

ouse gases in atmosphere that ultimately leads to global warming

1 , 2 ]. Biodiesel (monoalkyl esters of long chain fatty acids) is a po-

ential renewable biofuel and it is biodegradable, non-toxic, has no

et carbon dioxide and is free of sulfur [ 3 –6 ]. Generally, biodiesel is

roduced from food materials and oil crops using conventional meth-

ds [ 7 ]; however these sources cannot realistically replace the wide

se of diesel fuel due to increasing demand. Also the over population

orldwide has lead to serious land shortage and raised the issue of

ood security [ 8 ]. Microalgae have become a recent attraction because

f high oil content, and can be grown in wastewater as they do not

ompete with food crops for arable land and water and give 20 times

ore biomass productivity rate than the terrestrial crops [ 9 –14 ]. Mi-

roalgae are photosynthetic microorganisms that utilize light, water

nd CO 2 and accumulate intracellular lipids as storage materials [ 15 ].

Currently biodiesel is being produced employing conventional

ethods such as acid and alkali transesterification that results in the

onversion of triglycerides into fatty acid methyl esters in a shorter

eriod [ 16 , 17 ]. Major drawbacks of conventional methods include

igh energy input, elimination of salt, difficulty in recycling glycerol,

* Corresponding author.

E-mail address: suren micro@yahoo.co.in (D. Surendhiran).

213-3437/ $ - see front matter c © 2014 Elsevier Ltd. All rights reserved.

ttp://dx.doi.org/10.1016/j.jece.2014.05.004

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

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): NaNO 3 , 100;

NaH 2 PO 4 ·2H 2 O, 20.0; Na 2 EDTA, 4.0; H 3 BO 3 , 33.6; MnCl 2 ·4H 2 O, 0.36;

FeCl 3 ·6H 2 O, 13.0; vitamin B 12 , 0.001 and vitamin B 1 , 0.02. The trace

metal solution contained (g / l): ZnSO 4 ·7H 2 O, 4.4; CoCl 2 ·6H 2 O, 2.0;

(NH 4 ) 6 Mo 7 O 24 ·H 2 O, 0.9; and CuSO 4 ·5H 2 O, 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 saponificationand acid value of microalgal oil were determined. The molecularweight 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 mlscrew cap glass vial. No solvent was added in this reaction. The re-

action mixture consisted of 3 g of microalgal oil, 1 g of immobilizedwhole 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 smallamount of water at the concentration of 0, 2, 4, 6, 8, 10 and 12 wt% ofthe total amount of reaction mixture. The transesterification reaction

was allowed for 48 h at constant speed of 200 rpm. The biodiesel yieldwas calculated according to Umdu et al. [ 45 ]:

Biodiesel yield ( w t % ) =

(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.

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.

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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.

esults and discussion

uantification and characterization of microalgal oil

The oil content of C. salina was calculated according to Suganya

nd Renganathan [ 46 ] and the yield of oil extracted was found to be

8.26% (w / w). The lipid concentration was defined as dry weight ratio

f extracted lipids to biomass. The molecular weight of C. salina oil

as 849.88 (g / mol), which was calculated using acid value (0.42 mg

OH g −1 ) and saponification value (198.45 mg KOH g −1 ). The iodine

alue was found to be 65 (mg / g).

uantification of lipase assay

1% olive oil was used for enhancing lipase production. The activity

f lipase from the yeast R. mucilaginosa MTCC8737 was found to be

.26 U ml −1 .

ffect of biocatalyst loading

The cost of the enzyme involved in biodiesel generation affects the

verall economy of the production. Hence quantity of enzyme used

or the process should be mitigated [ 47 ]. In this study, effect of catalyst

as investigated for the interesterification reaction, and whole yeast

ells were used as biocatalyst adsorbed on sugar cane bagasse ( Fig.

). The surface area of sugar cane bagasse was found to be 1785 m

2 / g hich was analyzed using Mastersizer 2000, Malvern Instruments,

K. Effect of whole cell biocatalyst loading was studied to perform in-

eresterification in the range of 0.5 and 2.5 g. Fig. 2 showed that 1.5 g

f biocatalyst load yielded maximum biodiesel. The study revealed

hat higher cell concentration produced more biodiesel but above the

ptimal level of 1.5 g, the yield decreased. The result was in com-

lete agreement with Arumugam and Ponnusami [ 48 ]. This might be

ue to obstruction of mass transfer by larger particles. Moreover, the

uperfluous enzyme would unite and retard lipase activity [ 49 ].

ffect of oil and methyl acetate molar ratio

Oil to methyl acetate ratio is one of the key factors for biodiesel

roduction. The study showed that molar ratio 1:12 imparted largest

iodiesel yield of 56.2% at 48 h in the absence of solvents ( Fig. 3 ).

his report was consistent with Ognjanovic et al. [ 20 ]. As the mo-

ar ratio was raised to 1:14, a decline in production was observed.

he poor yield could be due to the presence of excess amount of

ethyl acetate that would dilute the oil [ 50 ]. Ratios lesser to the op-

imal value also exhibited insufficient yield. The conventional short

hains namely methanol or ethanol inactivated lipase above molar

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

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.

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.

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 ].

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

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 C 14 H 28 O 2 3.00

C16:0 Palmitic acid Hexadecanoic acid C 16 H 32 O 2 23.85

C16:1 Palmitoleic acid 9-Hexadecanoic acid C 16 H 30 O 2 11.18

C18:0 Stearic acid Octadecanoic acid C 18 H 36 O 2 9.55

C18:1 Oleic acid 9-Octadecenoic acid C 18 H 34 O 2 40.77

C18:2 Linoleic acid 9,12-Octadecadienoic acid C 18 H 32 O 2 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.

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Fig. 9. Gas chromatogram of FAME obtained from C. salina .

ne of the most advantageous phenomena of immobilized enzyme.

his is another major parameter influencing the overall production

conomy. Usually, stable and recyclable biocatalyst retards the cost of

eneration of biodiesel from oil [ 48 , 60 , 61 ]. This factor decides the pos-

ibility of large scale production of biodiesel utilizing enzymes [ 28 ].

n this section, stability and reusability of immobilized biocatalyst,

hole cells of R. mucilaginosa was investigated. The study revealed

hat there was no significant loss in activity of enzyme even after

tilizing after 10 cycles ( Fig. 8 ). This study was in contrast to that

f Srimhan et al. [ 3 ], which reported that yield, was decreased from

3.29 to 59.31 in the second cycle of bioconversion in the presence of

ethanol. But according to Du et al. [ 62 ], whose results were in agree-

ent to the present investigation showed that there was no loss of

iocatalyst even after 100 cycles of repeated usage in the presence

f methyl acetate. Methanol or ethanol, when used as acyl acceptor,

roduces glycerol as the byproduct, whose removal is intensive, and

ostly consuming and inactivates lipase. Thus, the present study in-

icated that immobilized lipase could be used for many cycles with

ethyl acetate as acyl acceptor that finally reduces the cost of overall

ioconversion process.

atty acid composition of C. salina FAME

Table 1 shows the six fatty acids present in the C. salina biodiesel.

rom the retention time obtained by GC-MS, peak values were ana-

yzed and observed as myristic acid (C14:0), palmitic acid (C16:0),

almitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1)

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

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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