Received:

25 May 2017
Revised:
Production of biodiesel by
14 October 2017
Accepted:
6 December 2017
enzymatic transesterification
Cite as: Arumugam A.,
Ponnusami V.. Production of
of waste sardine oil and
biodiesel by enzymatic
transesterification of waste
sardine oil and evaluation of its
evaluation of its engine
engine performance.
Heliyon 3 (2017) e00486.
doi: 10.1016/j.heliyon.2017.
performance
e00486

Arumugam A., Ponnusami V. *

School of Chemical and Biotechnology, SASTRA University, Thanjavur 613401, India

* Corresponding author.

Abstract

Waste sardine oil, a byproduct of fish industry, was employed as a low cost
feedstock for biodiesel production. It has relatively high free fatty acid (FFA)
content (32 mg KOH/g of oil). Lipase enzyme immobilized on activated carbon
was used as the catalyst for the transesterification reaction. Process variables viz.
reaction temperature, water content and oil to methanol molar ratio were
optimized. Optimum methanol to oil molar ratio, water content and temperature
were found to be 9:1, 10 v/v% and 30 °C respectively. Reusability of immobilized
lipase was studied and it was found after 5 cycles of reuse there was about 13%
drop in FAME yield. Engine performance of the produced biodiesel was studied in
a Variable Compression Engine and the results confirm that waste sardine oil is a
potential alternate and low-cost feedstock for biodiesel production.

Keywords: Environmental science, Chemical engineering

1. Introduction
The increase in environmental awareness and depletion of petroleum based diesel
had paved the way for the search of alternate fuels. Among several options
available, biodiesel is highly attractive and scalable. Biodiesel is preferred over

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 Article No~e00486

petroleum diesel as it can be derived from abundantly available vegetable/animal

sources. Moreover, emissions from combustion of biodiesel are relatively clean,
nontoxic and noncarcinogenic [1].

First generation biodiesels were mainly derived from edible plant and animal
sources. With increasing awareness on food security, second generation biodiesels
were produced from non-edible feedstock like non-edible vegetable oils, used
vegetable oils, and animal fats. Some of the second generation biodiesel
feedstocks, derived from plant sources, reported in the recent past include Castor
oil [2], Jatropha [3], Calophyllum inophyllum [4], Karanja [5], cotton seed [6],
neem [7], and used sun flower oil [8]. Similarly, animals wastes like beef tallow,
lard, and fish oil are low-value byproducts of meat and fishery industry and were
recently used as alternate feedstock for biodiesel production. Among them fish oil
is least explored as a raw material for biodiesel production. An enormous quantity
of waste is generated by fish industry, and indiscriminate disposal of these wastes
poses a threat to the environment. In 2014, world fish production was estimated to
be around 167 million tonnes [9]. Roughly about 50% of the total volume of fish
processed is discarded as waste during fish processing. Oil content in the waste
varies from 40% to 65% [10]. A recent survey reports that about 102,850 tons of
wastes are generated by fish processing industry located in Newfoundland and
Labrador alone, and this accounts for about 25% of the total fish waste generated in
Canada [11].

Generally, these wastes are used in low-value applications like animal feed [10]. In
some cases, where EicosaPentaenoic Acid (EPA) and DocosaHexaenoic Acid
(DHA) content of the oil is greater than 20%, these waste can also be used for the
recovery of a high-value product namely omega-3 concentrate which is used in
pharmaceutical and functional food applications. Ethyl esters of of eicosapentae-
noic acid and docosahexaenoic acid obtained by ethanolysis of sardine oil and tuna
oil were effective for the treatment of arteriosclerosis obliterans and hyperlipemia
[12, 13, 14]. However, EPA and DHA contents are usually very much lower than
20%, and therefore, recovery of omega-3 from fish oil is not economical. Thus,
most of the fish wastes are only used in low-value applications as indicated earlier.
In this scenario, use of fish oil waste for biodiesel production looks both
economically and environmentally attractive.

In canning industry, the mostly used oily fishes are sardine, mackerel, and tuna.
Processing of sardine, an important fish widely used in the commercial market,
also leads to discharge of considerable amount of wastes that include head, liver,
and intestine [15]. Waste sardine oil refers to the wastes which are collected during
canning process. Annual production of sardine oil was about 571,639 tons in the
year 2014 [16]. Reports on conversion of sardine oil into biodiesel are very much
limited as mentioned earlier. Therefore, the present work aims at conversion of

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 Article No~e00486

sardine oil into fatty acid methyl esters with potential application as biodiesel
through enzymatic transesterification.

Costa et al., 2013 utilized waste oil extracted from fish canning industry for
biodiesel production. The initial acid pre-treatment was done with sulfuric acid.
Biodiesel yield of 73.9 wt% was obtained by alkali catalyzed transesterification
with 1 wt. % catalyst and 60 vol. % of the methanolic solution [17].

Biodiesel is obtained from transesterification reaction involving a vegetable oil or

animal fat and a short-chain alcohol like methanol or ethanol in the presence of a
suitable catalyst (acid, base or biochemical catalyst). Compared to chemical
catalysts, enzymes require relatively low reaction temperature and less alcohol to
oil molar ratio. Also, chemical catalysts require multi-stage pretreatment if the
feedstock contains free fatty acids (FFA), particularly when FFA content is more
than 0.5 wt % of feedstock. Moreover, drawbacks such as product contamination,
wastewater generation, free fatty acids and water interferences and glycerol
recovery can be easily circumvented if enzyme catalysts are used. While using
enzyme catalysts, it is necessary to recover and reuse enzymes to reduce processing
cost. Immobilization of enzymes facilitates recovery and enables reuse of
expensive enzymes for several cycles [18]. Polyethylene gylated polyurathane
nanoparticles had been shown to be effective for immobilization of enzymes used
for esterification of fish oil [13, 14]. Immobilized lipase enzyme had been used to
catalyse ethanolysis of sardine oil and tuna oil [12, 13, 14].

This work is the first of its kind to report biodiesel production from waste sardine
oil catalyzed by lipase immobilized on activated carbon support matrix. Effect of
methanol to oil ratio (6:1–12:1), temperature (30 to 50 °C) and water content (5%
v/v − 15% v/v) were studied and optimized. Reusability of immobilized enzyme
was studied at 30 °C. Produced diesel was blended with petroleum diesel (10:90
ratio), and its performance and emission characteristics were evaluated using a
Variable compression ratio (VCR) engine.

2. Materials and methods

Waste sardine oil was collected from a canning industry located in Nagapattinam
District, Tamilnadu, India. The fatty acid profile of the collected sardine oil was as
follows: Octadecanoic acid (C18H36O2): 7.39%, Tridecanoic acid (C13H26O2):
15.54%, 9-Hexadecanoic acid (C 16 H 30 O 2 ): 13.47%, Hexadecanoic acid
(C 16 H 32 O 2 ):19.43%, 10-methyl Octadecanoic acid (C 19 H 38 O 2 ):19.58%,
5,8,11,14,17-Eicosapentaenoic acid (C20H30O2):16.08%, and Others: 8.5%.
Aspergillus niger Lipase (99%, 16 U/mg) and Methanol (99%) were purchased
from Himedia Laboratories Pvt. Ltd., India.

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2.1. Immobilization of lipase on activated carbon

The enzyme solution was prepared by in phosphate buffer at pH 7 to prevent the
deactivation of enzyme during immobilization [19]. Immobilized lipase was
obtained by mixing 150 ml of enzyme solution (protein content = 1 mg/ml; activity
= 16 U/ml) and 0.5 g of activated carbon. The above mixture was kept in a shaker
for 24 h at room temperature. After equilibration of the mixture, the solution was
filtered using Whatman filter paper, and immobilized lipase was collected
separately [20]. Percentage immobilization was estimated as follows (Eq. (1)):
S0  St
Percentageimmobilization ¼ × 100 (1)
S0

Where S0 and St refer to protein concentration in the solution before and after
immobilization. Protein concentration was determined using Lowry’s method. The
specific enzyme activity of immobilized lipase was determined using olive oil
emulsion method [21].

2.2. Biodiesel production

Biodiesel was produced as described by Arumugam et al., 2014 [4]. Briefly, waste
sardine oil was mixed with methanol (methanol to oil ratio 6:1, 9:1 and 12:1) and
water (5, 10 and 15% v/v). Immobilized Aspergillus niger lipase (100 mg) was
added to the mixture, and the reaction mixture was maintained at a constant
temperature (30 to 50 °C) in an orbital shaker at 200 RPM for 10 h [22]. Reaction
time also affects the yield of biodiesel. Typical reaction time for enzymatic
transesterification of various oils range from 4 h to 72 h [23, 24]. Our preliminary
experiments on transesterifcation of sardine oil, with Aspergillus niger lipase (free
enzyme) as catalysts, suggested that maximum conversion could be achieved in
less than 10 h reaction time. Thus, in this study with immobilized lipase enzyme,
we have chosen 10 h reaction time.

2.3. Properties of biodiesel

The properties of biodiesel- such as Cetane number, Cloud point, Flash point,
Density, Kinematic viscosity were determined using ASTM methods [25]. The
composition of the biodiesel produced was estimated by Gas Chromatography −
Mass spectroscopy (GC − MS) (Clarus 500, PerkinElmer, USA).

2.4. Experimental setup for engine testing

Specification of the Variable Compression Ratio (VCR) engine used in this study
is given in Table 1. For engine test, diesel − biodiesel (90:10) blended fuel (B10)
was used. Fuel injection pressure and compression ratio (CR) were maintained at
200 bar and 18:1 respectively during the test run. The flue gas from engine exhaust

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Table 1. VCR engine specifications.

Engine type Kirloskar/PS 234

Number of cylinder One

Stroke length (mm) 110
Bore (mm) 87.5
Swept volume (cc) 661.45
Compression ratio 12:1 to 18:1
Engine speed (rpm) 1500
Injection timing (°CA) 20−40
Injection opening pressure (bar) 210

was analyzed with DELTA 1600-L (specification is shown in Table 2). The engine
was started with no load, and the required speed and steady state condition were
attained by adjusting the feed control. Data acquisition system was used to measure
power output, rpm, exhaust gas temperature, and fuel consumption.

3. Results and discussion

3.1. Percentage immobilization and specific enzyme activity
The percentage lipase immobilization and specific enzyme activity were found to
be 57% and 13 U/mg of support matrix, respectively. The specific enzyme activity
of the immobilized enzyme was similar to the free enzyme (16 U/mg), showing
that there was no reduction in specific enzyme activity due to the matrix − enzyme
interaction during the immobilization process.

3.2. Biodiesel production

As mentioned in materials and methods section, immobilized lipase enzyme was
used to produce biodiesel from sardine oil. Methanol to oil ratio, percentage water
content and temperature are the most important process variables that affect

Table 2. Gas analyzer specifications.

Measured quantity Measuring Range Resolution

O 0-15.00% 0.01%
CO2 0-20.00% 0.01%
Hydrocarbons 0–20000 ppm 1 ppm
NOX 0–2000 ppm 1 ppm

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biodiesel yield. Optimum values of these variables in turn depend on the biodiesel
feedstock used, source of lipase and type of immobilization. Therefore, effect of
these variables of biodiesel yield was studied.

3.2.1. Effect of methanol-to-oil ratio

It is well known that both esterification and transesterification reactions are
equilibrium-limited. To shift the reaction towards ester formation, excess methanol
is usually used [26]. Also, excess alcohol minimizes diffusion limitations by
reducing the viscosity of reaction mixture and improves mass transfer rate and in
turn enhances overall reaction rate. In the present study, we used 6:1, 9:1, and 12:1
methanol to oil molar ratios to study the effect of methanol to oil ratio on the yield
of biodiesel. The results are shown in Fig. 1(a). Biodiesel yield increased from
85.73% to 94.5% (p < 0.001) when methanol to oil molar ratio was increased from
6:1 to 9:1. Further increase in the molar ratio did not promote ester formation.
Thus, for further studies methanol to oil molar ratio 9:1 was used. Optimum
alcohol to oil ratio reported in previous literature widely varies. It varies from 3.5
to 12, depending upon biodiesel feedstock, microbial source of enzyme and
molecular weight of alcohol used in the reaction [27]. Too much of excess
methanol could result in emulsification of glycerol and reduce biodiesel yield

[(Fig._1)TD$IG] 6:1 5%
(a) 100
9:1
(b) 10%
12:1 100 15%
FAME Percentage Yield

FAME Percentage Yield

80 80

60 60

40 40

20 20

0 0
6:1 9:1 12:1 5% 10% 15%
Methanol: oil (molar) Water content (v/v%)

(c) 100
25
(d) 100
30
FAME Percentage Yield

FAME Percentage Yield

80 35 80
40

60 60

40 40

20 20

0 0
25 30 35 40 1 2 3 4 5
Temperature oC Number of cycles

Fig. 1. (a) Effect of methanol to oil molar ratio (6:1, 9:1, 12:1) on methanolysis of waste sardine oil for
temperature 30 °C and reaction time of 10 h (b) Effect of percentage water content (5%, 10%, 15%) on
methanolysis of waste sardine oil for 9:1 molar ratio of methanol to oil, temperature 30 °C, and reaction
time of 10 h (c) Effect of temperature (T = 25, 30, 35 and 40 °C) on methanolysis of waste sardine oil
for methanol to oil molar ratio of 9:1 and reaction time of 10 h (d) Reusability studies Immobilized
lipase on methanolysis of waste sardine oil at optimum conditions.

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through recombination of ester and glycerol [28]. Also use of excess methanol, a
polar short chain linear alcohol, beyond a certain level may lead to structural
changes in lipase and cause enzyme deactivation [29]. Various approaches had
been followed by previous researchers to reduce enzyme denaturation. This
includes: (i) stepwise addition of methanol (ii) use of other acyl acceptors like
ethanol, 2-butanol, 2-propanol, methyl acetate, ethyl acetate, etc. as alternate to
methanol and (iii) use of organic solvents. But, all of these approaches have their
own advantages and disadvantages [18]. Overall, simple single step addition of
methanol has been most commonly accepted by researchers as best option.

3.2.2. Effect of water percentage on methanolysis

In transesterification reaction catalyzed by enzymes water plays a vital role in the
conversion of triglycerides to Fatty Acid Methyl Esters (FAME) [30] as the
stability and catalytic activity of the lipase enzyme are affected by the presence of
water/any other solvent. Lipase requires an oil-water interface for their action.
Addition of water favors formation of oil-water droplets and hence increase
interfacial area available for the catalytic action. Also, dispersing the lipase in
water/solvent prevents deactivation of lipase by methanol [31]. The effect of water
content on the enzyme-catalyzed transesterification reaction of waste sardine oil
was investigated with different percentage of water (5%, 10%, and 15%) and the
results are shown in Fig. 1(b). The highest conversion of approximately 92.5% was
obtained at a water content of 10% (v/v). Apart from transesterification reaction,
lipase also catalyzes other reactions like hydrolysis. Presence of excess water
favors hydrolysis and hence lowers biodiesel results [32].

3.2.3. Effect of temperature

Temperature is another important factor influencing reaction rate. Rate of reaction
increases with increase in temperature. However, for an enzymatic reaction at
higher temperatures reaction rate falls beyond some point due to enzyme
deactivation. Optimum temperature for an enzyme catalyzed transesterification
reaction depends on enzyme as well as alcohol used. For most of the lipase
catalyzed transesterification reactions, optimum temperature falls in the range of
30 to 50 °C [30]. We have studied the effect of temperature on methyl ester yield
by increasing reaction temperature from 25 at an interval of 5 °C, until there was a
noticeable drop in the FAME yield. The results are shown in Fig. 1(c). The FAME
yield was significantly higher (p < 0.001) at 30 °C and further experiments were
conducted at 30 °C.

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3.2.4. Reusability
The reusability for the lipase immobilized on activated carbon was studied for five
cycles. From Fig. 1(d), it can be seen that FAME yield decreased very slowly upon
each successive transesterification cycle. After five successive cycles, there was
about 13% reduction in FAME yield. The percentage reduction in biodiesel yield
was likely due to leaching of the lipase from the support matrix [32].

Enzymes can be immobilized on support matrix by chemical or/and physical

methods. Chemical methods include adsorption, ionic binding and covalent
bonding. Physical methods include entrapment and encapsulation. Each method
has their own advantage and disadvantage. Adsorption involves only weak forces,
enzyme leaching is a common problem associated with this method. Inspite of this
shortfall, adsorption is still one of the most popular methods of enzyme
immobilization owing its inherent advantages over other immobilization methods.
Some of the advantages of this method include (i) simple, (ii) does not require
toxic chemical treatment, and (ii) mass transfer resistances (particularly internal)
are neglible. Ionic binding and covalent bonding methods employ stronger
chemical bonding between enzyme and support matrix. As the binding between the
enzyme and matrix is stronger enzyme leaching can be either completely prevented
or minimized. However, it may cause a drop in the enzyme activity if the some of
the active sites of the enzymes are blocked in the process. Moreover, this method
requires chemical treatment of the support matrix/enzyme prior to immobilization.
Physical methods like entrapment and encapsulation can also prevent leaching of
the enzyme. However, strong internal mass transfer resistances becomes a
limitation in these methods and result in lower reaction rate. Most recently, hybrid
methods involving combination of two or more immobilization methods had been
shown to be effective in certain cases. In this study, we have used only adsorption
for enzyme immobilization and the results are in favor of the method. However, the
optimum/best method of immobilization for any enzyme-substrate system can be
established only after examing all methods with careful consideration of synthesis
cost, enzyme reusability, enzyme stability, ease of product separation, etc. [23].

3.2.5. Properties of biodiesel

The fuel properties of biodiesel produced from waste sardine oil are listed in
Table 3. All these properties except for cloud point were well within the ASTM
standards for diesel fuel. A comparison some of the enzymes catalyzed
transesterification processes for the biodiesel production, reported in recent
literature, is given in Table 4.

Cloud point of biodiesel (16 °C) was higher than for a diesel fuel. This is a cause
for concern, particularly, if the biodiesel is to be used in countries where the
ambient temperature is below its operability limits, leading to potential cold start

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Table 3. The comparison of biodiesel production from various sources using

immobilized lipase on different support matrix in the literature with the present
work.

Properties of biodiesel Sardine oil biodiesel ASTM methods and of biodiesel

Density (kg/m3) at 15 °C 891 D5002-94 (860–900)

Flash point (°C) 161 D93 (130)
Cloud point (°C) 16 D91 (-3 to 12)
Kinematic viscosity at 40 °C (cSt) 3.9 D445 (1.9 to 6)
Cetane number 56 D613 (49)

problems. However, use of additives, preheating and filtration before injection can
be useful to overcome this issue and to improve low-temperature operation of
biodiesel. Thus, sardine oil could be an alternative feedstock for biodiesel
production. Ester content of the biodiesel produced was found to be: 9-
Hexadecenoic acid, methyl ester, (Z)- (C17H32O2) − 13.2%; Hexadecanoic acid,
methyl ester (C17H34O2) − 19.41%; Tridecanoic acid, methyl ester (C14H28O2) −
14.95%; 5,8,11,14,17-Eicosapentaenoic acid, methyl ester (C21H34O2) − 16.25%;
10-methyl octadecenoic acid, methyl ester: (C20H40O2) − 19.13%; Octadecanoic
acid, methyl ester: (C19H38O2) − 10.49%.

3.3. Performance and emission characteristics

3.3.1. Brake thermal efficiency
Brake Thermal Efficiency is defined as the ratio of the heat equivalent of the break
output to the heat supplied to the engine. It is used to evaluate the effectiveness of
an engine in converting the heat from combustion of fuel to mechanical energy
[47]. About 10–12% of oxygen present in vegetable oil based fuel can favorably
affect combustion efficiency of biodiesel as compared to petroleum diesel [48].
That is, the ester molecules present in the biodiesel favors complete combustion.
The biodiesel blend has brake thermal efficiency (BTE) lower than that of diesel
fuel in all percentage load conditions (Fig. 2(a)). The BTE values for B10 and
diesel fuel were 27% and 33% respectively indicating that the diesel fuel has a
higher BTE value at 100% load [49]. Lower BTE of the biodiesel blend compared
to diesel fuel is mainly due to high fuel density, high viscosity and low heating
value of the blends [3].

3.3.2. Specific fuel consumption (SFC)

The Specific Fuel Consumptions (SFC) of B10 blend was compared with
petroleum diesel, and the results are shown in Fig. 2(b). Biodiesel SFC was higher

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Table 4. The comparison of biodiesel production from various sources using immobilized lipase on different support matrix in the literature with the present
work.

Reference Support Source Catalyst/Enzyme Reaction %

time yield

Jegannathan et al. 2010 [33] κ-carrageenan Palm oil lipase PS from Burkholderia 72 h 100.0
cepacia
Juan Camilo Naranjoa et al. Activated carbon Palm oil Candida 12 h 100.0
2010 [34] antarctica B lipase
Jingjing Zheng et al. 2012 [35] K2SO4 micro-crystals Soybean oil, sunflower seed oil, olive oil, camellia oil, corn oil and Pseudomonas cepacia lipase 12 h 99.8
rapeseed oil
Yao Wang 2014 [36] Acrylic resin crude algal oils Novozym 435 4h 99.1
Huang Y et al. 2010 [37] Resin Lard oil Novozym 435 and Lipozyme 20 h 97.6
TLIM
Kaili Nie et al. 2011 [38] Textile fibre Salad oil and Waste oil Candida sp. 99–125 lipase 30 h 96.0
Li et al. 2010 [39] Micro-aqueous phase Sapium sebiferum oil Lipase from 12 h 97.1
Pseudomonas cepacia G63
Present work Activated carbon Sardine oil Lipase from Aspergillus niger 10 h 94.0
Yucel, 2011 [40] Olive pomace Pomace oil Lipase from Thermomyces 24 h 93.0
lanuginosus
Kawakami et al, 2011 [41] Silica monolith Jatropha oil Burkholderia cepacia lipase 12 h 90.0
Huang et al, 2015 [42] Crystalline PVA microspheres Microalgae Rhizomucor miehei lipase 24 h 90.0
Oil
Yan et al. 2010 [43] Cross-linking and lipase coating with Waste cooking oil Geotrichum sp. Lipase 4h 85.0
K2SO4
Kalantari et al. 2013 [44] Magnetic silica nanocomposite Soybean oil lipase from Pseudomonas cepa- 24 h 55.0
cia
Roman et al. 2017 [45] Chitosan Coconut oil Pseudomonas fluorescens lipase 24 h 12.0

Article No~e00486
Ramani Kandasamy et al. 2010 Activated carbon Olive oil Pseudomonas gessardii 2h -
[46]
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[(Fig._2)TD$IG]
40

Specific fuel consumption(kg/kW)

1.0
BTE - BD SFC - BD

Break thermal efficiency (%)

BTE - DF SFC - DF
0.8
30

0.6
20
0.4

10
0.2

0 0.0
20 40 60 80 100
Percentage engine load

Fig. 2. Performance characteristics of sardine oil biodiesel. (BTE - Break thermal efficiency; SFC -
Specific fuel consumption; BD - 10% Biodiesel blend; DF - Petroleum based diesel fuel) (a) Variation
in specific fuel consumption with load for diesel fuel and B10. (b) Variation in brake thermal efficiency
with load for diesel fuel and B10.

compared to petroleum diesel. High viscosity, density and low heat content of
biodiesel blend when compared with that of diesel attribute to the high SFC. The
presence of additional oxygen molecules in the fuel ensures better/complete
combustion of the fuel. This is considered to be the major reason for higher SFC
and higher power generation [50]. The ignition performance of the fuel is also
improved due to oxygenated nature of the biodiesel. Cetane number was greater
than that of diesel fuel [51].

[(Fig._3)TD$IG]
50 6000
CO2 (ppm)
CO (ppm)
HC (ppm)
Concentration (ppm)

40 NOx (ppm)
Concentration, ppm

4000
30

20
2000

10

0 0
Biodiesel Diesel fuel
Fuel

Fig. 3. Emission characteristics of biodiesel compared with petroleum diesel. (a) Variation in CO2 and
CO emission at 100% load for diesel fuel and biodiesel. (b) Variation in HC and NOX emission with
100% load for diesel fuel and biodiesel.

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3.3.3. Exhaust gas emission

A five gas analyzer (AVL Di Gas − 4000 model) was used to analyze exhaust
discharges such as carbon monoxide, nitrogen oxides, and unburned hydrocarbon
(Fig. 3). Carbon emission is based on the efficiency of combustion and the fuel's
carbon content, which will undergo a series of oxidation reactions during
combustion. The complete oxidation of carbon leads to the formation of CO2. If the
oxidation is not complete, the exhaust will contain more of CO and hydrocarbons
[52].

The formation of CO was 22.2% lower at 100% load in biodiesel due to the
presence of oxygen atoms as mentioned earlier [53]. However, higher oxygen
content of the fuel results in a reduction in the calorific value in biodiesels. Fig. 3
shows that biodiesel (B10) emits less amount of hydrocarbon compared to diesel
fuel (36% reduction). The partially or completely burnt hydrocarbons are found in
the emissions from the engine [54]. Once again this is attributed the presence of
oxygen molecules in the fuel [55].

NOX are generated in a diesel engine because of high flame temperature, peak
pressure prevailing inside the cylinder, nitrogen content of the fuel and the
residence time of the fuel inside the cylinder [56]. Biodiesel shows 25% reduction
in NOx emissions compared to diesel fuel, which is essentially due to the reduction
of temperature in the combustion chamber. Higher the temperature, greater is the
amount of NOx emission. The residence time or ignition delay may be less in ester-
based fuel because of its high cetane number [55]. The delay period for the ester is
reduced because of the presence of oxygen which increases the ignition quality.

4. Conclusion
Biodiesel was produced from waste sardine oil, a non-edible renewable source, by
transesterification catalyzed by lipase immobilized on activated carbon. The
optimal conditions for methanolysis are 9:1 (mol/mol) methanol to oil ratio, 10%
water content and 30 °C. Under these optimal conditions, about 94.55% of methyl
ester yield was obtained. Immobilized enzyme could be used for 5 cycles without
appreciable loss in activity. Specific energy consumption and exhaust emissions
also saw substantial advantages in biodiesel as compared with diesel fuel. The
performance analysis of 10% blend of waste sardine biodiesel with diesel fuel
showed that sardine oil biodiesel used along with petroleum fuel as a blend
improves the engine performance and minimizes emission of air pollutants in the
flue gas. Use of such blended fuel does not require any key hardware alteration to
the structure.

12 http://dx.doi.org/10.1016/j.heliyon.2017.e00486
2405-8440/© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
 Article No~e00486

Declarations
Author contribution statement
A. Arumugam: Conceived and designed the experiments; Performed the
experiments; Analyzed and interpreted the data; Contributed reagents, materials,
analysis tools or data; Wrote the paper.

V. Ponnusami: Analyzed and interpreted the data; Contributed reagents, materials,

analysis tools or data; Wrote the paper.

Funding statement
This research did not receive any specific grant from funding agencies in the
public, commercial, or not-for-profit sectors.

Competing interest statement

The authors declare no conflict of interest.

Additional information
No additional information is available for this paper.

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