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Yasvanthrajan 2020
Yasvanthrajan 2020
Yasvanthrajan 2020
PII: S2352-1864(20)31499-1
DOI: https://doi.org/10.1016/j.eti.2020.101199
Reference: ETI 101199
Please cite this article as: N. Yasvanthrajan, P. Sivakumar, K. Muthukumar et al., Production of
biodiesel from waste bio-oil through ultrasound assisted transesterification using immobilized
lipase. Environmental Technology & Innovation (2020), doi:
https://doi.org/10.1016/j.eti.2020.101199.
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Thanabalan Murugesan3, Appusamy Arunagiri1*
1
Department of Chemical Engineering, National Institute of Technology, Tiruchirappalli,
Tamil Nadu, India, 620015.
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2
School of Petroleum Technology, Pandit Deendayal Petroleum University, Gandhinagar,
Gujarat, India, 382007.
3
Department of Chemical Engineering, University Teknologi Petronas, Perak, Malaysia
*
Corresponding author Email id: aagiri@nitt.edu
Abstract
In this study, process intensification was carried out using lipase catalyzed
transesterification of waste cottonseed oil (WCSO) with ethanol in the presence of ultrasonication.
A maximum conversion of 98.7% was observed for ultrasound assisted transesterification. The
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optimum process conditions were ethanol to oil molar ratio of 4.5:1, a reaction temperature of
45°C, enzyme loading of 5 wt%, a reaction time of 6 h, ultrasonic amplitude of 40% and the duty
cycle of 15 s ON and 15 s OFF. The influence of ultrasound makes the process efficient because
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of an effective reduction in reaction time to 6 h with the least catalyst dosage and power
consumption. Reusability tests of the catalyst were conducted after separating the immobilized
Rhizopus oryzae lipase. The regenerated lipase catalyst was found to have better reusability up to
the fourth cycle and the produced biodiesel was within the ASTM D6751 standard. Furthermore,
the ultrasound assisted process under mild operating conditions did not affect the immobilized
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enzyme. It was clearly observed that ultrasonication is faster and effective for biodiesel conversion
using the immobilized lipase catalyzed transesterification reactions.
1. Introduction
Energy is the key to economic growth and the industrial affluence of a nation. The energy
sector around the globe is reliant on fossil fuels leading to global warming that poses one of the
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biggest threats to mankind. It has given a wake-up call to both the developed and developing
countries who are on the quest for alternative fuels (Kothari et al., 2020). The gradual progression
of green fuels like biodiesel has the potential to address the energy crisis and to replace the fossil
fuels without the need to make major changes in the existing diesel engines (Jindapon et al., 2020).
Biodiesel that are produced from renewable sources like edible and non-edible oils and fats can
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be comfortably used in compression ignition engines. Biodiesel can be produced in the presence
or absence of a catalyst and the process can be both chemical and biochemical. The catalysts are
selected based on the quality of the feedstock. During the reaction, oil reacts with acyl acceptors
like alcohols to give biodiesel and glycerol as by-products (Amini et al., 2017). Among the
different types of catalysts used, enzymes are preferred as they can catalyze low grade feedstock
having high Free Fatty Acid (FFA) and water. Enzymes such as Rhizopus oryzae, Thermomyces
lanuginosus, Candida antarctica, Pseudomonas fluorescens, Pseudomonas cepacia, Rhizomucor
miehei etc. produced from different microbial strains having high catalytic activity to carryout
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alcohols such as ethanol, butanol, etc. (Christopher et al., 2014). Furthermore, the process can also
be enhanced by intensification approaches such as microchannel reactors, microwave and
ultrasonic assisted enzymatic biodiesel synthesis which reduces not only the reaction time but also
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enzyme inhibition. Many researches have been carried out in the above discussed intensification
process with efficient conversion. Among them, ultrasonication is found to have four times the
shorter reaction time for effective conversion (Natarajan et al., 2019; Thangarasu et al., 2020; Yan
et al., 2014).
Ultrasound waves are generated using bath and probe type instruments. During
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ultrasonication, fluid tends to undergo rapid distortion due to the compression and rarefaction
movement initiated by the sonic waves. This is followed by a cavitation effect on the reaction
stream formed by the rapid creation and collapse of micro bubbles (Ho et al., 2016). This process
significantly affects the mechanism of the reaction thereby enhancing the reaction rate.
Consecutively, non-uniformity in the cavitation effect greatly reduces the overall performance of
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the reaction which can be avoided by appropriate reactor designs and the careful selection of
operating conditions. Hence, the impact of ultrasound can increase the catalyst activity by its least
contribution to the reaction and it also has the tendency to increase the mass transfer rates (Luo et
al., 2014).
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Ultrasonic assisted enzymatic biodiesel synthesis factually reduces the reaction time from
24 to 4 h by increasing the interfacial area that ultimately reduces the catalyst quantity, reaction
time and temperature (Ho et al., 2016). Though several researches have been carried out in enzyme
applications for biodiesel production, the literature pertaining to ultrasound assisted enzymatic
transesterification process is limited. Therefore, the combined application of enzyme and
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ultrasound has shown a reduction in reaction time and increase in catalytic activity under mild
operating conditions. Ultrasound irradiation generates turbulence resulting in the increase in the
mass transfer rate that dominates the process of intensification in biodiesel production using
commercially immobilized lipase (Bansode and Rathod, 2017). Hence the combined application
of enzyme and ultrasound has shown a reduction in reaction time and increase in catalytic activity
under mild operating conditions.
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The chief limitation in the commercial use of traditional biodiesel is its cost factor. The
high cost is mainly due to the selection of raw materials, catalyst consumption and reusability,
design of the reactor and the overall operation cost. Among them, the selection of the raw materials
is found to be the most important parameter that contributes to the process economy up to 75-
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80%. Biodiesel is priced more than diesel and the high cost can be curtailed by the utilization of
cost effective feedstock derived from waste biomass sources such as non-edible oils, algae, waste
cooking oil, waste fats, waste lipids, industrial wastes, etc. since enzymes can catalyze feedstock
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with varying FFA (Berrios et al., 2011; Gude et al., 2013). In this study, Waste cottonseed oil
(WCSO) was chosen as the raw material to reduce the production cost. The transesterification
reaction was catalyzed by immobilized Rhizopus oryzae lipase. The influence of the various
process parameters such as ethanol to oil molar ratio, reaction time, temperature, catalyst loading,
ultrasonic amplitude and duty cycle were investigated. Furthermore, reusability tests were
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conducted to the study the action of the enzyme under ultrasonication.
lipase immobilized on Immobead 150, from Rhizopus oryzae (≥300 U/g) was obtained from
Sigma Aldrich, Bangalore, India. It was stored in refrigerator (2-8°C) as instructed by the material
data sheet. Ethanol, used as a reactant in this experiment was of analytical grade (purity of 99%)
that was obtained from Merck, Mumbai, India.
Ultrasound assisted transesterification was carried out in a 500 mL glass reactor having a
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double wall jacketed column for water circulation in order to maintain a desired temperature
between 35 and 55°C. The schematic representation of the ultrasonic reactor is shown in Figure
1. A probe type ultrasonic processor (13 mm dia) (VCX 750, Sonics and Material Inc, USA) with
a maximum operating power of 750 W and a frequency of 20 kHz was inserted into the glass
reactor. The ultrasonic probe was fixed in the reaction mixture in such a way that it did not touch
either the bottom or the walls of the reactor to assure uniform irradiation on the reaction mixture.
The amplitude of the ultrasonicator was varied between 20% and 100%. Similarly, the duty cycle
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was changed by varying the consecutive duty cycle of 5 s ON and 5 s OFF to 30 s ON and 30 s
OFF. During the reaction, the ethanol to oil molar ratio and the catalyst loading was varied
between 3:1 and 6:1 from 2 to 6 wt% respectively. Initially, the oil was charged into the reactor
and heated to the desired temperature and then the enzyme along with ethanol was added to the
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reaction stream. The temperature was carefully maintained by circulating the water using the
constant temperature water bath throughout the reaction. On the completion of the reaction, the
product was collected and separated in a high-volume ultra-centrifuge after which it was
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transferred into a separating vessel for two phase separation. Biodiesel was formed as the top layer
whereas glycerol along with the catalyst, the bottom layer. After the separation of the upper layer,
vacuum evaporator was used to remove the excess unreacted ethanol. Reusability tests were
conducted after the enzymes were washed with benzyl acetate to obtain the maximum possible
regeneration of the catalyst. Apart from this, no buffer solution or water was added to the reaction
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mixture.
Enzyme assay was estimated using WCSO as the substrate and it was incubated with the
immobilized enzyme in phosphate buffer. When the incubation time was completed, the mixture
was titrated with NaOH in the presence of phenolpthalin as an indicator (Subhedar and Gogate,
2016). Immobilized enzyme activity was expressed as µmole of the ester obtained per minute and
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the activity percentage was calculated as given in eq (1).
ୡ୲୧୴୧୲୷ ୭ ୰ୣୱ୦ ୧୫୫୭ୠ୧୪୧ୣୢ ୣ୬୷୫ୣ
Enzyme Activity ሺ%ሻ = × 100 (1)
ୡ୲୧୴୧୲୷ ୭ ୣ୬୷୫ୣ ୳ୱୣୢ ୧୬ ୣୟୡ୦ ୡ୷ୡ୪ୣ
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The conversion of WCSO into biodiesel was estimated using H1NMR spectroscopy, Bruker
AVANCE III 500 MHz. The protons adjacent to the ester moiety in triglyceride and the protons
in the alcohol moiety of the product were used to calculate the conversion of oil to biodiesel
(Jaiswal et al., 2016; Silva et al., 2012). The presence of biodiesel is also confirmed by the FT-IR
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spectra from its functional group. Biodiesel was analysed by PerkinElmer spectrum 100
(PerkinElmer Inc. USA) having the interpretation value of wave number between 400 and 4000
cm-1. The physical and chemical properties of the biodiesel were analysed according to ASTM
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standard methods and compared with ASTM D6751 standard.
3. Results and Discussions
Property Values
Density at 15°C (kg m-3) 0.912 ±0.1
Viscosity at 40°C (mm2 s-1) 0.93 ±0.1
Avg. molecular weight (g mol-1) 868.13 ±2
Saponification value (mg KOH g-1 oil) 197.6 ±2
Acid value (mg KOH g-1 oil) 1.31 ±0.2
Percentage FFA (%) 4.565 ±0.5
Iodine number (g I2(100 g)-1 ) 103.24 ±2
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The fatty acid profile of WCSO is given in Table 2. The average molecular weight of
WCSO is found to be 868.13 g mol-1 with 31.25% of saturated and 68.45% of unsaturated fatty
esters. Furthermore, the mass ratio of the saturated and the unsaturated fatty acids is 0.45 which
suggests that the produced fuel will be comparatively less viscous. This results in better fluidity
and engine operation by reducing the engine deposits. However, high unsaturation leads to least
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oxidation stability due to carbon chain length and presence of large number of double and triple
bonds in the fatty chain compounds. Hence, it requires an anti-oxidant to increase its storage
stability. Therefore, WCSO can be considered as a potential raw material for biodiesel production.
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Fatty acid Oil (wt%)
Myristic acid 0.92
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Palmitic acid 24.6
Palmitoleic acid 0.6
Stearic acid 5.1
Oleic acid 14.85
Linoleic acid 53.2
Linolenic acid 0.4
Undetectables 0.33
The effect of ethanol to oil molar ratio in the reaction has been found to be between 3:1 and
6:1 with an increase in 0.5 moles of ethanol while the other parameters were kept constant for the
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reaction with and without ultrasound. The results obtained for the different ethanol to oil molar
ratios are shown in Figure 2. It has been observed that the conversion is minimum at 3:1 molar
ratio which gradually increases with the increase in ethanol to oil molar ratio. The same trend has
been observed for reactions with and without ultrasound. In both the cases, the biodiesel
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conversion has been found to be maximum at 4.5 ethanol to oil molar ratio and with a further
increase in the molar ratio, the conversion starts decreasing. Thus, ultrasound increases the
conversion percentage of oil to biodiesel by enhancing the reaction rate thereby achieving the
stoichiometry in lower molar ratio when compared to the conventional process. This is due to the
ultrasound irradiation which favours mass transfer rate between the immiscible phases of the
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The maximum conversion of 81% and 43% were obtained at 4.5:1 ethanol to oil molar ratio
which is considered as the optimum in both the cases. This clearly suggests that the presence of
ultrasound improves conversion by 53% when compared to conventional process. Addition of
ethanol to the reaction stream did not alter the conversion and furthermore, it was observed that
the addition of excess ethanol negatively impacted the reaction because of its potential to inhibit
the enzyme catalyzed reaction (Subhedar and Gogate, 2016). The cavitation bubbles created by
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the ultrasound generated fine emulsion between the immiscible reactants whereby the interfacial
area between the reactants was enhanced which in turn led to a reduction in ethanol consumption
and ultimately resulting in a boost to the reaction rate (Kalva et al., 2009).
95
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With ultrasound Without ultrasound
85
75
Conversion (%)
65
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55
45
35
25
15 re-
2.5 3 3.5 4 4.5 5 5.5 6
Ethanol molar ratio
Figure 2. Effect of ethanol to oil molar ratio vs conversion at temperature 40°C; reaction time of
8 h; 4 wt% enzyme concentration; amplitude of 60% and duty cycle of 10 s ON and 10 s OFF)
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3.2.2 Effect of temperature:
The temperature was one of the most important parameters for carrying out the reaction
and it was specially taken care of for better conversion rates. The effect of reaction temperature
ranging from 35 to 55°C with an increment of 5°C rise, was studied in the presence and absence
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of ultrasound. The reaction temperature was optimized using the results shown in Figure 3. When
the operating temperature was kept at 35°C, biodiesel conversion was 54.6% and 34.7% for with
and without ultrasound respectively is not efficient due to poor mass transfer limitations i.e. the
minimum temperature required to perform the collision between the enzyme and substrate to form
the complex to enact the reaction is not sufficient (Yadav and Jadhav, 2005). When the
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temperature was raised to 40°C, the reaction proceeded further with better increment in conversion
attaining a maximum at 45°C with 86.5% biodiesel conversion. With temperatures above 45°C,
the conversion started to decrease which is attributed to the fact that enzymes are heat sensitive
and above a particular temperature, the weak molecular forces between the ionic structure and
support are damaged invariably affecting the conversion (Subhedar and Gogate, 2016). Biodiesel
conversion is found to be maximum at 45°C for both the cases, i.e. 86.5 and 52.6 %. When the
temperature is below 40°C, it does not increase the conversion which is due to the poor miscibility
and weak interactions between the molecules of the reactants and this leads to a decrease in the
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conversion (Arumugam and Ponnusami, 2017; Subhedar et al., 2015). The mass transfer between
the reactants will be more realistic when the temperature is at 45°C. The conversion reaches
optimum when the mass transfer between the reactants is high.
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95 Without ultrasound With ultrasound
85
75
Conversion (%)
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65
55
45
35
25
15
30 35 40
re- 45
Temperature ( °C)
50 55
Figure 3. Effect of temperature vs conversion at ethanol to oil molar ratio of 4.5:1; reaction time
of 8 h; enzyme concentration of 4 wt%; amplitude of 60%; duty cycle of 10 s ON and 10 s OFF)
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3.2.3 Effect of catalyst loading:
The quantity of the catalyst consumed in the process determines the production cost.
To study this impact, the catalyst quantity was varied from 2 to 6 wt% by keeping the other
parameters constant. The obtained results are shown in Figure 4. The data obtained for the lowest
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enzyme loading of 2 wt% has the lowest conversion of 48.6% which indicates that the lower
concentration of enzyme lacks the efficiency to catalyze the reaction stream. On the other hand,
when the concentration is increased from 2.5 to 4.5%, there is an increasing pattern in the
conversion of 90.2% and 61.2% for the reaction with and without ultrasound respectively. The
same trend was observed by (Kumar et al., 2011) while using the immobilized lipase catalyst. A
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further increase in enzyme loading above 5wt% did not show much difference in the conversion
from where the conversion began to fall slightly. The higher concentration above this showed
decreasing pattern and the catalyst variation in this range did not have an effective influence on
the conversion. This may be due to the agglomeration of the catalyst dispersed in the reaction
medium which in turn reduces the contact between the active sites and the reactants (Zhao et al.,
2015). Hence the optimized catalyst loading is found to be 5 wt%.
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Conversion (%) 90
75
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60
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45
30
1 2 3 4 5 6
Enzyme loading (wt%)
Figure 4. Effect of enzyme loading vs conversion at ethanol to oil molar ratio of 4.5:1; reaction
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temperature of 45°C; reaction time of 8 h; amplitude of 60% and ultrasonic duty cycle of 10 S ON
and 10 S OFF)
Reaction time is the most crucial parameter when enzymes are involved because they
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require longer time when compared to other chemical catalysts such as NaOH and KOH.
Moreover, the use of ultrasound considerably decreases the reaction time. It generates cavitation
resulting in the formation of a large number of micro bubbles which then collapses to increase the
mass transfer of the reaction (Bansode and Rathod, 2017). Using this process, the reaction time
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for the enzyme catalyzed reaction with and without ultrasound is optimized by keeping the others
parameters constant. The results obtained are shown in Figure 5. Initially, the reactions were
started with a minimum reaction time of 2 h and gradually the reaction time was increased to 14
h and the conversions were monitored. The maximum conversion reported was 92.2% at 6 h of
reaction time and an increase in the reaction time above this did not have any impact on the
conversion. This is due to the fact that the reaction attained its equilibrium whereby it did not
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contribute to any additional improvement in the conversion. Moreover, the rate of forward reaction
is equal to the reverse reaction during this time (Christopher et al., 2014). The curve is found to
be similar in both cases from 4.5 to 6 h with little variations in the conversion and the maximum
conversion is achieved at 6 h. (Subhedar and Gogate, 2016) obtained the maximum yield with
lesser time in 3 h which might be due to difference in the feedstock selection. However, prolonged
exposure of ultrasound has no deviations until 6 h. These observations clearly suggest that
sonication process can reduce the time effectively to 6 h, whereas, the reaction without ultrasound
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did not reach the equilibrium even after 14 h (71.9%) and the maximum conversion is less than
with ultrasound condition. Hence, the optimized reaction time was 6 h in ultrasound assisted
transesterification process.
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100
Without ultrasound With ultrasound
90
Conversion (%)
80
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70
60
50
40 re-
0 2 4 6 8 10 12 14
Reaction time (h)
Figure 5. Effect of reaction time vs conversion at ethanol to oil molar ratio of 4.5:1; reaction
temperature of 45°C; enzyme loading of 5 wt%; ultrasonic amplitude of 60%; ultrasonic duty
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cycle of 10 S ON and 10 S OFF)
The influence of ultrasonic irradiation causes dispersion in the reaction mixture due to the
cavitation effect and this helps in obtaining the mixing patterns of the immiscible substrates. The
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ultrasound amplitude was varied between 20% and 100% in intervals of 20% while the other
parameters were kept constant. The obtained results are shown in Figure 6. During the ultrasonic
process intensification, the rapid breakdown of the micro bubbles during adiabatic compression
generates temperature and pressure spots resulting in the production of shockwaves that
simultaneously leading to the increase of both mass and heat transfers in the reaction (Ho et al.,
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2016). The bubbles created in the medium are in accordance with the power amplitude and the
precise amplitude results in higher biodiesel conversion (Bansode and Rathod, 2017). The reaction
proceeded with 20% amplitude has the maximum conversion of 85% due to the degree of
intensification that causes the dissipation of cavitational bubbles created inside the reaction stream
to achieve the internal mass transfer within the immiscible phase of the reactants. When the
amplitude was increased to 40%, there was an acute rise of 93.8 % in the biodiesel conversion.
An increase in the ultrasonic amplitude beyond 40 % was found to have a slight decrease in the
conversion and when the amplitude was kept at 80 and 100%, the conversion rate decreased
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rapidly due to the collapse of the bubbles causing disruption which in turn affected the conversion.
Better mass transfer rate is achieved only when the amplitude is kept at the minimum because an
increase in amplitude causes increased cavitational intensity leading to enzyme inhibition
(Waghmare et al., 2015).
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100
95
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Conversion (%)
90
85
80
75
0 20 40
re- 60 80 100
Amplitude (%)
Figure 6. Effect of ultrasonic amplitude vs conversion at ethanol to oil molar ratio of 4.5:1;
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reaction temperature of 45°C; reaction time of 6 h; catalyst loading of 5 wt%; ultrasonic duty cycle
of 10 S ON and 10 S OFF)
Ultrasonic duty cycle is an important step that governs the economics of the reaction
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by controlling the process conversion and the equanimity of the enzyme immobilized reaction. In
a nutshell, duty cycle is the amount of sonication time used for varying the intervals to enhance
the reaction. Duty cycle also reduces the leaching of enzyme in the immobilized medium. It takes
place in the reaction to decrease the possibility of enzyme deactivation since the duty cycle is
alternately controlled to supply ultrasonic irradiation (Adewale et al., 2015). The performance and
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optimization of ultrasonic irradiation was studied by changing the duty cycle from 5 s ON and 5
s OFF to 30 s ON and 30 s OFF. The highest possible duty cycles above 30 s and up to 50 s ON
and 50 s OFF have not been taken for analogy since they could possibly damage enzyme
immobilization and lead to wear and tear problems (Waghmare et al., 2015). The effect of duty
cycles for varying ultrasonic irradiations is shown in Figure 7. The observed results show that a
duty cycle of 5 s ON and 5 s OFF has a conversion rate of 75% and with an increase in the duty
cycle to 10 s ON and 10 s OFF has a maximum conversion of 93.8%. The highest conversion of
98.7% is obtained with a duty cycle of 15 s ON and 15 s OFF. Moreover, the rise in the duty cycle
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from 20 s to 30 s ON and OFF shows a substantial decrease in the yield to 74.2% which is because
of the extended exposure time of enzymes in the reaction medium (Waghmare et al., 2015).
Furthermore, an increase in the duty cycle does not enhance the conversion; rather it leads to a
decrease in the conversion which is due to the reaction mechanism of lipase catalyzed
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transesterification using ultrasonication (Adewale et al., 2016). Enzyme immobilization has
distinct reaction mechanisms compared to chemical catalyst. It actually requires a minimum
reaction time for the catalyst to allow the triglycerides react with the surface functionalized active
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sites and allow the substrates to convert the reaction following its pathway (Medina et al., 2009).
The optimization parameters of duty cycle conclude that the minimum of 15 s ON and 15 s OFF
is the most effective duty cycle at which the enzyme attains the minimum permissible time to react
with the substrates thereby eliminating the chances of enzyme deactivation. In addition to this, the
optimized duty cycle effectively reduces energy consumption and avoids over heating of the
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process.
100
Conversion (%)
90
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80
70
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60
0 5 10 15 20 25 30 35
Duty cycle (s)
Figure7. Effect of ultrasonic duty cycle vs conversion at ethanol molar ratio of 4.5:1; reaction
temperature of 45°C; enzyme loading of 5 wt%; reaction time of 6 h; ultrasonic amplitude of 40%)
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FT-IR spectra of the biodiesel and cottonseed oil are shown in Figure 8. The presence
of ester peak at 1744 cm-1 and C-H peak near 2750-3125 cm-1 confirms the presence of fatty acid
ester group. The C-O bond in the range of 1000-1250 cm-1 present in the ester group can be
observed as more than one peak. In accordance with GC results, the peak at 1745 cm-1 confirms
the presence of oil having highly unsaturated fatty acids which is shown by peak C=C bonds and
also the presence of linoleic acid, linolenic acid and other unsaturated acid ethyl ester as observed
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from the peak at a characteristic of C‚C stretching. The presence of functional groups in cottonseed
oil and biodiesel shows similar spectral features without much difference and they contain C=O,
O-CH3, -CH3, O-CH2, C-H and CH=CH. The conversion of cottonseed oil to biodiesel was
confirmed by the characterized absorption peaks of C=O stretching. The shift at carbonyl group
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of the biodiesel from 1156 to 1170 cm-1 also confirms the presence of biodiesel. Similarly, C=O
stretching peaks appear at 2923, 2973 and 2854 cm-1. The =C-H stretching is observed at wave
number 600–850 cm-1.
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100
95
2973
Transmittance %
723
90 1433
2854
85
re- 1745
1170
1156
80 2923
75
1733
70
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Oil Biodiesel
65 800
600
400
4000
3800
3600
3400
3200
3000
2800
2600
2400
2200
2000
1800
1600
1400
1200
1000
To ensure the quality of the biodiesel produced from WCSO, the fuel properties were
analysed using ASTM methods. The values obtained were compared with ASTM D6751 standard
which were found to be in accordance with the limits. The results given in Table 3 show that the
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viscosity of the biodiesel is found to be 4.59 mm2 s-1 which proves that the fuel has longer liquid
penetration and invariably affects the atomization properties. It also determines lubricity which in
turn affects the burning efficiency (Choksi et al., 2019). The flash and fire point of the fuel are
161 and 166°C which clearly indicate that it can be stored and transported safely. Similarly, the
oxidative stability is 9 h which confirms the good keeping quality of the fuel (Booramurthy et al.,
2020). The cetane index is 51.88 which implies a better fuel ignition quality. The total sulphated
ash is found to be 0.4 which clearly states that engine corrosion will be minimum. Moreover, the
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amount of water and sediments found in the fuel is 0.39 which shows that engine stoppage and
the cause of microbial contamination in the fuel is negligible. Furthermore, the percentage of
carbon residue is found to be 0.26 which will reduce fuel injector fouling and cylinder scoring
(Arumugamurthy et al., 2019). Sodium and Potassium, Calcium and Magnesium were below 5
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ppm. Hence, the fuel produced will be safe for use in internal combustion engines.
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Properties Methods ASTM D6751 Biodiesel
Viscosity (mm2 s-1) D445 1.9-6 4.59 ± 0.3
Acid value (mg KOH g-1) D664 0.5 max 0.39 ± 0.01
Flash point (°C) D93 93 min 161 ± 2
Fire point (°C) D93 - 166 ± 2
Cloud point(°C) D2500 - 4.5 ± 0.3
Pour point(°C) D97 - 2 ± 0.2
Cetane index EN ISO 4264 -
re- 51.88 ± 2
Sulfated ash (% mass) D874 0.02 max 0.01 ± 0.01
Water and sediments (vol %) D2709 0.050 max 0.03 ± 0.01
Carbon residue (% mass) D524 0.5 max 0.26 ± 0.01
Na and K combined (ppm) EN14538 5 max 1.2 ± 0.1
Ca and Mg combined (ppm) EN14538 5 max 2.6 ± 0.1
Oxidation stability (h) EN14112 8 min 9 ± 0.5
Min and max represent minimum and maximum respectively*
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operating cost. With these possible considerations, enzymes are immobilized whereby enzyme
stability is enhanced for specific enzymes. If the enzymes are reused without regeneration, the
enzyme activity decreases whereby it affects conversion. This is due to the leaching of enzymes
and reduction in activity (Guldhe et al., 2015). After the reaction, the active sites in the enzymes
have to be regenerated to retain the catalytic activity by reactivating the possible active sites. After
the completion of the reaction, the enzyme was separated and washed using benzyl acetate. The
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solvents have been chosen carefully to avoid possible inhibition and to favour the enhancement
of enzyme activity. Washing was done thrice to remove the residues leftover and the unreacted oil
and ethanol. The immobilized enzymes were then dried using vacuum drier for 15 min at room
temperature and preserved in a desiccator at preferred storage temperature of 8°C. Thereafter, the
immobilized lipase was rinsed twice with 15 mL Tris-HCl buffer (pH 8.5) at 20°C for 4 h to
regenerate lipase activity (Yu et al., 2013).
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The used enzymes were tested for six cycles and the obtained results are represented in
Figure 9. The catalytic activity retained till the fourth cycle is appreciable with a conversion above
80% and biodiesel satisfies the ASTM standard. After the fourth cycle, the produced biodiesel did
not match the standard and also the enzyme activity decreased. In the fifth and sixth cycles,
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conversion and enzyme activity decreased to a greater extent. A similar pattern result was obtained
by various researchers and a comparative study was carried out. Michelin et al., (2015) has
explained the reusability of Novozym 435 for the kinetics of macauba coconut oil using ultrasonic
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assisted transesterification and have observed a decrease in conversion after every cycle which is
due to the reduction in enzyme activity. It has been stated that ultrasound does not specifically
affect enzyme stability and activity, rather it has been found that ultrasound induces the reaction
rate by enhancing the catalyst activity to obtain the highest yield by optimization (Michelin et al.,
2015). Waghmare et al., (2015) have studied ultrasound assisted bath process using Novozym
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435 since it has no catalyst reusability for six cycles (Waghmare et al., 2015). In addition to this,
Subhedar and Gogate (2016) have reported that the enzyme reusability of Lipozyme using
ultrasound assisted intensification resulted in decreased biodiesel yield when the enzymes were
reused after the second cycle (Subhedar and Gogate, 2016). Several studies have reported that
enzyme activity decreases resulting in a decrease in the conversion. The possibility of increasing
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the catalyst reusability by using the chemical activation of the immobilized Rhizopus oryzae is
explored in this study that has shown an appreciable increase in conversion. The linear reduction
in enzyme activity followed by the decrease in biodiesel conversion is due to the denaturation of
the enzyme which occurs as the result of the collapse of the enzyme along with the carrier support
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as the practical constraints lead to the leaching of enzymes and this affects the efficiency of the
process.
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120
Conversion (%) Catalyst activity (%)
100
100 95 92 95
87 90
82 84
Percentage (%)
80 75.4 74
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68.4 67
60
40
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20
0
1 2 3 4 5 6
Number of cycles
4. Conclusion
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Figure 9. Reusability of enzymes at optimum reaction conditions.
The present work studied the ultrasound assisted transesterification of WCSO using
immobilized Rhizopus oryzae. The effect of process parameters such as ethanol to oil molar ratio,
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catalyst loading, reaction temperature, time, ultrasonic amplitude and duty cycle were studied and
the conventional experimental results were compared with ultrasound assisted process. The results
proved that the impact of ultrasound on the catalytic activity of the immobilized Rhizopus oryzae
lipase greatly reduced the reaction time and catalyst consumption that helped in alleviating the
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When it is extended to industrial scale, it will reduce the overall energy consumption and reward
with better return on investments.
Acknowledgements:
The authors would like to express their gratitude to National Institute of Technology,
Tiruchirappalli, India under the Ministry of Human Resource Development, Govt. of India for
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providing a platform to carry out research and fellowship to Mr. Natarajan Yasvanthrajan
(NITT/Ph.D./402115051).
References:
of
Adewale, P., Dumont, M.J., Ngadi, M., 2016. Enzyme-catalyzed synthesis and kinetics of
ultrasonic assisted methanolysis of waste lard for biodiesel production. Chem. Eng. J. 284,
158–165.
pro
Adewale, P., Dumont, M.J., Ngadi, M., 2015. Enzyme-catalyzed synthesis and kinetics of
ultrasonic-assisted biodiesel production from waste tallow. Ultrason. Sonochem. 27, 1–9.
Amini, Z., Ilham, Z., Ong, H.C., Mazaheri, H., Chen, W.H., 2017. State of the art and
prospective of lipase-catalyzed transesterification reaction for biodiesel production.
Energy Convers. Manag. 141, 339–353. re-
Arumugam, A., Ponnusami, V., 2017. Production of biodiesel by enzymatic transesterification
of waste sardine oil and evaluation of its engine performance. Heliyon e00486.
Arumugamurthy, S.S., Sivanandi, P., Pandian, S., Choksi, H., Subramanian, D., 2019.
Conversion of a low value industrial waste into biodiesel using a catalyst derived from
lP
brewery waste : An activation and deactivation kinetic study. Waste Manag. 100, 318–
326.
Bart, J.C.J., Palmeri, N., Cavallaro, S., 2010. Emerging new energy crops for biodiesel
production, in: Bart, J.C.J., Palmeri, N., Cavallaro, S. (Eds.), Biodiesel Science and
Technology From Soil to Oil. Woodhead Publishing, pp. 226–284.
Berrios, M., Martín, M.A., Chica, A.F., Martín, A., 2011. Purification of biodiesel from used
cooking oils. Appl. Energy 88, 3625–3631.
Jou
Bharathiraja, B., Chakravarthy, M., Kumar, R.R., Yuvaraj, D., Jayamuthunagai, J., Kumar,
R.P., Palani, S., 2014. Biodiesel production using chemical and biological methods – A
review of process, catalyst, acyl acceptor, source and process variables. Renew. Sustain.
Energy Rev. 38, 368–382.
Booramurthy, V.K., Kasimani, R., Subramanian, D., Pandian, S., 2020. Production of biodiesel
from tannery waste using a stable and recyclable nano-catalyst : An optimization and
Journal Pre-proof
Choksi, H., Pandian, S., Arumugamurthi, S.S., Sivanandi, P., Sircar, A., Booramurthy, V.K.,
2019. Production of biodiesel from high free fatty acid feedstock using heterogeneous acid
catalyst derived from palm-fruit-bunch. Energy Sources, Part A Recover. Util. Environ.
of
Eff. 1–10.
Christopher, L.P., Kumar, H., Zambare, V.P., 2014. Enzymatic biodiesel: Challenges and
pro
opportunities. Appl. Energy 119, 497–520.
Gude, V.G., Grant, G.E., Patil, P.D., Deng, S., 2013. Biodiesel production from low cost and
renewable feedstock. Cent. Eur. J. Eng. 3, 595–605.
Guldhe, A., Singh, B., Mutanda, T., Permaul, K., Bux, F., 2015. Advances in synthesis of
biodiesel via enzyme catalysis : Novel and sustainable approaches. Renew. Sustain.
re-
Energy Rev. 41, 1447–1464.
Ho, W.W.S., Ng, H.K., Gan, S., 2016. Advances in ultrasound-assisted transesterification for
biodiesel production. Appl. Therm. Eng. 100, 553–563.
Jaiswal, S.K., Prakash, N.T., Prakash, R., 2016. H NMR Based Quantification of Ethyl Ester
lP
in Biodiesel : A Comparative Study of Product- Dependent Derivations. Anal. Chem. Lett.
6, 518–525.
Jindapon, W., Ashokkumar, V., Rashid, U., 2020. Production of biodiesel over waste seashell-
derived active and stable extrudate catalysts in a fixed-bed reactor. Environ. Technol.
rna
Kalva, A., Sivasankar, T., Moholkar, V.S., 2009. Physical Mechanism of Ultrasound-Assisted
Synthesis of Biodiesel. Ind. Eng. Chem. Res. 48, 534–544.
Kothari, R., Vathistha, A., Singh, H.M., Pathak, V.V., Tyagi, V.V., Yadav, B.C., Ashokkumar,
V., Singh, D.P., 2020. Assessment of Indian bioenergy policy for sustainable environment
Jou
and its impact for rural India: Strategic implementation and challenges. Environ. Technol.
Innov. 20, 101078.
Kumar, G., Kumar, D., Poonam, Johari, R., Singh, C.P., 2011. Enzymatic transesterification of
Jatropha curcas oil assisted by ultrasonication. Ultrason. Sonochem. 18, 923–927.
Luo, J., Fang, Z., Jr, R.L.S., 2014. Ultrasound-enhanced conversion of biomass to biofuels.
Prog. Energy Combust. Sci. 41, 56–93.
Journal Pre-proof
Medina, A.R., Moreno, P.A.., Cerdan, L.., Grima, E.M., 2009. Biocatalysis : Towards ever
greener biodiesel production. Biotechnol. Adv. 27, 398–408.
Michelin, S., Penha, F.M., Sychoski, M.M., Scherer, R.P., Treichel, H., Valerio, A., Luccio,
M.D., Oliveira, D.D., Oliveira, J.., 2015. Kinetics of ultrasound-assisted enzymatic
of
biodiesel production from Macauba coconut oil. Renew. Energy 76, 388–393.
Natarajan, Y., Nabera, A., Salike, S., Tamilkkuricil, V.D., Pandian, S., Karuppan, M.,
pro
Appusamy, A., 2019. An overview on the process intensification of microchannel reactors
for biodiesel production. Chem. Eng. Process. Process Intensif. 136, 163–176.
Silva, G.A.M., Ros, P.C.M.D., Souza, L.T.A., Costa, A.P.O., Castro, H.F.D., 2012. Physico-
chemical, spectroscopical and thermal characterization of biodiesel obtained by enzymatic
route as as tool to select the most efficient immobilised lipase. Brazilian J. Chem. Eng.
29, 39–47.
re-
Subhedar, P.B., Botelho, C., Ribeiro, A., Castro, R., Pereira, M.A., Gogate, P.R., Paulo, M.A.,
2015. Ultrasound intensification suppresses the need of methanol excess during the
biodiesel production with Lipozyme TL-IM. Ultrason. Sonochem. 27, 530–535.
lP
Subhedar, P.B., Gogate, P.R., 2016. Ultrasound assisted intensification of biodiesel production
using enzymatic interesterification. Ultrason. Sonochem. 29, 67–75.
Thangarasu, V., Siddharth, R., Ramanathan, A., 2020. Modeling of process intensification of
biodiesel production from Aegle Marmelos Correa seed oil using microreactor assisted
rna
Waghmare, G.V., Vetal, M.D., Rathod, V.K., 2015. Ultrasound assisted enzyme catalyzed
synthesis of glycerol carbonate from glycerol and dimethyl carbonate. Ultrason.
Sonochem. 22, 311–316.
Yadav, G.D., Jadhav, S.R., 2005. Synthesis of reusable lipases by immobilization on hexagonal
Jou
Yan, Y., Li, X., Wang, G., Gui, X., Li, G., Su, F., Wang, X., Liu, T., 2014. Biotechnological
preparation of biodiesel and its high-valued derivatives : A review. Appl. Energy 113,
1614–1631.
Yu, X., Sha, C., Guo, Y., Xiao, R., Xu, Y., 2013. High-level expression and characterization
of a chimeric lipase from Rhizopus oryzae for biodiesel production. Biotechnol. Biofuels
Journal Pre-proof
6, 1–12.
Zhao, X., Qi, F., Yuan, C., Du, W., Liu, D., 2015. Lipase-catalyzed process for biodiesel
production : Enzyme immobilization, process simulation and optimization. Renew.
Sustain. Energy Rev. 44, 182–197.
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Highlights
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Maximum conversion of 98.7% is achieved at the optimum conditions
Process optimization studies and reusability of the catalyst are carried out
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Pandian Sivakumar, Karuppan Muthukumar and Thanabalan Murugesan: Writing –
Review and Editing, Supervision, Resources
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Declaration of interests
The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work
reported in this paper.
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The authors declare the following financial interests/personal relationships
which may be considered as potential competing interests:
Corresponding author:
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Dr.A.Arunagiri,
Associate Professor,
Fax: +91-431-2500133
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Corresponding author Email id: aagiri@nitt.edu
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