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Ultrasound assisted intensification of biodiesel production using enzymatic interesterification
Accepted Manuscript Ultrasound assisted intensification of biodiesel production using enzymatic interesterification Preeti B. Subhedar, Parag R. Gogate PII: DOI: Reference:
S1350-4177(15)30027-4 http://dx.doi.org/10.1016/j.ultsonch.2015.09.006 ULTSON 2990
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
30 June 2015 5 September 2015 6 September 2015
Please cite this article as: P.B. Subhedar, P.R. Gogate, Ultrasound assisted intensification of biodiesel production using enzymatic interesterification, Ultrasonics Sonochemistry (2015), doi: http://dx.doi.org/10.1016/j.ultsonch. 2015.09.006
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Ultrasound assisted intensification of biodiesel production using enzymatic
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interesterification
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Preeti B. Subhedar, Parag R. Gogate*
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Chemical Engineering Department,
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Institute of Chemical Technology,
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Matunga, Mumbai – 400 019, India
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*
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Tel.: +91 22 33612024,
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Fax: +91 22 33611020;
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Corresponding author
E-mail address: [email protected]
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Abstract
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Ultrasound assisted intensification of synthesis of biodiesel from waste cooking oil
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using methyl acetate and immobilized lipase obtained from Thermomyces lanuginosus
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(Lipozyme TLIM) as a catalyst has been investigated in the present work. The reaction has
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also been investigated using the conventional approach based on stirring so as to establish the
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beneficial effects obtained due to the use of ultrasound. Effect of operating conditions such as
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reactant molar ratio (oil and methyl acetate), temperature and enzyme loading on the yield of
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biodiesel has been investigated. Optimum conditions for the conventional approach (without
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ultrasound) were established as reactant molar ratio of 1:12 (oil : methyl acetate), enzyme
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loading of 6% (w/v), temperature of 40 ºC and reaction time of 24 h and under these
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conditions, 90.1% biodiesel yield was obtained. The optimum conditions for the ultrasound
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assisted approach were oil to methyl acetate molar ratio of 1:9, enzyme loading of 3% (w/v),
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and reaction time of 3 h and the biodiesel yield obtained under these conditions was 96.1%.
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Use of ultrasound resulted in significant reduction in the reaction time with higher yields and
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lower requirement of the enzyme loading. The obtained results have clearly established that
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ultrasound assisted interesterification was a fast and efficient approach for biodiesel
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production giving significant benefits, which can help in reducing the costs of production.
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Reusability studies for the enzyme were also performed but it was observed that reuse of the
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catalyst under the optimum experimental condition resulted in reduced enzyme activity and
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biodiesel yield.
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Key words: Ultrasound, Intensification, Interesterification, Biodiesel, Immobilized Lipase,
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Operating parameters
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1. Introduction
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Biodiesel offers a substitute to the petroleum based fuels offering greener processing and
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reduced emissions. Biodiesel (monoalkyl fatty acid methyl esters), which is generally
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produced by the transesterification reaction of triglycerides with methanol, has become
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significantly important in recent years due to the diminishing petroleum reserves, strong
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dependence of the national economies on the fuel prices and the ecological concerns of
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released gases from petroleum-based fuel [1]. Even though biodiesel has been generally
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produced using the chemical synthesis approach, there are numerous problems associated
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with this approach such as recovery of glycerol, removal of inorganic salts and significant
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processing costs especially with the separation of excess methanol being used in the synthesis
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[2, 3].
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Enzyme catalyzed approach for production of biodiesel partially addresses these issues and
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offers an environmentally more attractive alternative to the conventional processes [4-6]. The
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enzyme catalyzed biodiesel production under moderate environments allows the easy
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removal of the biocatalyst and glycerol recovery by centrifugation resulting in achieving
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biodiesel production with high purity in simple steps [7]. Typically short-chain alcohols such
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as methanol are used as the acyl acceptor for biodiesel production, however, usage of
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methanol in excess leads to deactivation of enzyme, and glycerol which is a major by-product
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of the reaction, also has inhibitory effects on the enzymatic activity apart from offering low
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commercial value. These associated issues with the enzymatic route have offered
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considerable restrictions for the effective application at industrial scale [8]. Replacing the
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alcohol as alkyl acceptor by alkyl acetate helps to solve these problems due to the use of
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alcohol and the reaction is described as interesterification.
3
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The present work deals with production of biodiesel using the interesterification reaction
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based on methyl acetate as an acyl acceptor instead of methanol and the by-product formed in
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the reaction is triacetin instead of glycerol (Scheme 1). Triacetin has higher commercial value
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as compared to glycerol due to the use as a gelatinizing agent as well as additives in tobacco,
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pharmaceutical and cosmetic industries [9]. Moreover, triacetin has no adverse effect on the
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activity of lipase enzyme [10]. Though interesterification offers significant advantages, very
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few papers associated to this reaction route have been reported. Kim et al. [11] used ethyl
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acetate as acyl acceptor and highest biodiesel production yield of 63.3% was achieved by
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using an ethyl acetate to oil molar ratio of 6:1 molar ratio with 8% of the immobilized lipase
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Novozym 435.
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synthesis of biodiesel based on the use of methyl acetate instead of more commonly used
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alcohol. Maximum biodiesel yield of 92.34% was obtained by using 1.5 g of enzyme bead,
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1:12 molar ratio of oil to methyl acetate, temperature of 35 °C in 60 h of reaction time.
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Apart from possible inactivation of enzyme, another key shortcoming in the
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commercialization of biodiesel production is the cost of operation especially dominated by
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the higher costs of the enzyme, raw materials and significantly longer reaction times. Even
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though, biodiesel has an enormous potential to substitute exhaustible fossil fuel, for all the
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current processing technologies, the cost of biodiesel is about 50 to 100% higher than the
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petroleum based diesel fuel [13]. The significant cost associated with the raw materials [14]
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can be reduced by using waste cooking oil (WCO) or other sustainable resources. The mass
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transfer limitations due to the heterogeneous nature of reactants can be reduced based on the
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use of intensification approaches such as using ultrasound. Use of interesterification approach
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instead of transesterification can also help in producing a more valuable co-product with the
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biodiesel. Considering these aspects, interesterification of waste cooking oil using enzymatic
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route and intensification using ultrasound has been investigated in the present work.
Surendhiran and Vijay [12] studied the interesterification reaction for
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Application of ultrasound can give substantial degree of process intensification based on
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cavitation phenomenon. The cavitational effects can improve the mass transfer at mild
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reaction conditions in terms of temperature and pressure resulting into faster reaction rate,
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higher product yield [15, 16] and possibly requirement of lower acyl acceptor to oil molar
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ratio and catalyst loading. Cavitation creates intense turbulence and liquid circulation at
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micro scale which help in reducing the mass transfer resistances in heterogeneous systems
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[17]. Although researchers have used ultrasound for the enhancement of transesterification
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route for biodiesel synthesis [18-20], there is practically no information available for its
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application for the enzymatic interesterification route of biodiesel production. The main aim
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of the present work was to investigate the influence of ultrasound on the enzymatic
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interesterification of waste cooking oil using immobilized lipase. Furthermore, different
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reaction conditions including molar ratio of substrates, enzyme loading, reaction temperature
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and time which might influence the yield of FAMEs have been investigated so as to
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maximize the yield. Comparison of the ultrasound based approach with the conventional
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approach has been presented so as to establish the benefits of using ultrasound as a source of
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mixing.
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2. Materials and methods
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2.1. Materials
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Waste cooking oil (WCO) was obtained from a local restaurant in Mumbai, India. The initial
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analysis of oil revealed that the main contents were unsaturated fatty acids (linoleic and oleic
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acids as a total of 91%) with very less quantum of saturated fatty acids (palmitic and stearic
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acid). The properties of WCO used in the work for the biodiesel production are shown in
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Table 1. Methyl acetate was acquired from S.D. Fine Chemicals Ltd., Mumbai. Acetonitrile 5
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and acetone (HPLC grade) used as HPLC solvents were obtained from J.T. Baker, Mumbai.
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The methyl oleate and methyl linoleate standards were procured from Sigma-Aldrich. Lipase
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enzyme, Lipozyme TL IM, immobilized on silica granules, was kindly provided as a gift
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sample by Brenntag India Pvt. Ltd., Mumbai, India.
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2.2.2. Interesterification of waste cooking oil
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A 100 mL glass reactor equipped with mechanical stirrer and baffles to avoid vortex
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formation was used for the conventional approach of interesterification. The temperature of
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the reaction was maintained constant using water bath. Reactor was also equipped with a
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condenser to achieve complete reflux conditions and recycle the methyl acetate vapours back
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to the reaction mixture. WCO and methyl acetate were first fed to the reactor and heating was
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started. After reaching the desired temperature, lipase enzyme was added into the reactor. 0.5
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ml samples were withdrawn at specific intervals and analysed using HPLC. It was observed
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that the pH of the reaction mixture did not change with time. The effect of operating
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parameters such as reactant molar ratio, temperature and enzyme loading has also been
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investigated. The reproducibility of the data was tested by performing the experiments in
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duplicate and average values have been reported. The observed errors were within 2% of the
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reported average value.
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2.2.3. Ultrasound assisted interesterification of waste cooking oil
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Ultrasonic horn obtained from Dakshin, Mumbai was used for the approach of ultrasound-
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assisted enzymatic hydrolysis. The ultrasonic irradiation at a frequency of 20 kHz was
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transferred into the reaction mixture through a titanium cylindrical horn, submerged 2.0 cm
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into reaction mixture. Ultrasonic horn had a diameter of 1.1 cm and maximum rated power
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output of 120 W. The schematic representation of experimental set up has been shown in Fig.
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1. The procedure followed for the ultrasound-assisted enzymatic interesterification was 6
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similar to that mentioned in the conventional approach of enzymatic interesterification but the
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samples were withdrawn after 30 min for methyl ester analysis. The temperature and flow
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rate of circulating water bath was maintained in such a way that the reaction temperature was
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always controlled to 50 °C. The effect of different parameters such as ultrasonic power,
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ultrasonic duty cycle, oil to methyl acetate molar ratio and enzyme loading has been
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investigated.
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2.3. Analytical methods
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2.3.1. Acid value
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The acid value of the waste cooking oil was determined by the acid-base titration technique
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as per the details mentioned in our earlier work [9].
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2.3.2. Fatty acid composition and fatty acid methyl ester analysis
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The original composition of the fatty acids in the oil were determined by converting all the
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fatty acids into corresponding esters and subsequently analyzed using gas chromatography
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(GC) based on the use of BP-X70 column. The progress of the reaction was monitored in
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terms of the fatty acid methyl ester content using Agilent Eclipse XDB C-18 HPLC column,
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having dimensions of 4.6 × 250 mm. Acetonitrile: acetone (70:30 v/v ratio) was used as a
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mobile phase and samples were analyzed isocratically at a flow rate of 1.5 mL/min. Samples
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for analysis were prepared by diluting 10µL of the reaction mixture in 10 mL of mobile
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phase. The concentration was determined based on the method of calculating the area under
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the peak obtained at the specific retention time.
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2.3.4. Enzyme assay
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The enzyme assay was done using tributyrin as a substrate for enzyme. Tributyrin (0.2 mL)
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was incubated with the lipase enzyme (200 mg) in phosphate buffer (pH 7) for 5 min. After 5
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min of incubation, the reaction was ended by the addition of methanol (20 mL) and reaction
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mixture was titrated against alcoholic NaOH (0.1 M) using phenolphthalein as an indicator.
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Immobilized enzyme activity can be expressed as µmoles of ester formed per minute and was
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calculated using the following equation:
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Enzyme activity (TBU/g) =
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where V = volume in ml of NaOH which is a measure of tributyrin consumed during
167
reaction, M = molarity of NaOH, E = amount of enzyme employed in mg and T = time of
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reaction in min. One unit of lipase activity was defined as the amount of enzyme required to
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hydrolyze 1 mole of ester bond per minute under assay conditions.
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2.3.4. Structural characterisation of immobilised lipase
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Circular dichroism (CD) spectroscopy was used to monitor the secondary structure of the
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non-sonicated and ultrasound irradiated immobilised lipase enzyme. The CD spectra were
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recorded on a JASCO J-810 CD instrument (JASCO). CD spectra were scanned in the far UV
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range (200 to 250 nm) with three replicates at 50 nm/min with bandwidth of 0.5 nm. Cell
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length was 10 mm. In all measurements, the lipase concentration was kept constant at 50
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mg/mL. The CD data were expressed in terms of ellipticity in mdeg. All the spectra were
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corrected by subtracting a blank spectrum (without lipase).
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2.3.5. Environmental scanning electron microscopy (ESEM)
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To understand the effect of ultrasonic irradiation on the immobilized lipase, samples were
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analyzed by environmental scanning electron microscopy. The analysis of the enzyme was
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performed before and after the ultrasonic irradiation. Samples were analyzed by Quanta 200
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unit (FEI, Field Emission Instruments, Hillsboro, OR) at 20 kV for determination of the
V × M × 1000 W× T
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morphological changes occurred due to the use of ultrasound. Samples of immobilized
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lipases were displaced in metallic plates and submitted to a previous treatment of atomization
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with gold to improve the image definition and characteristics.
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3. Results and discussion
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3.1 Effect of reactant molar ratio
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In the interesterification reaction, one mole of triglycerides reacts with three moles of methyl
190
acetate giving one mole of biodiesel, indicating a stochiometric requirement of oil to methyl
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acetate molar ratio (OMAMR) as 1:3. An excess of methyl acetate is typically used to drive
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the reaction in the forward direction. Experiments were performed with OMAMR ranging
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from 1:6 to 1:15. Fig. 2(a) depicts the obtained results for the conventional approach of
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stirring, where it can be seen that an increase in the molar ratio from 1:6 to 1:12 results in an
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increase in the biodiesel yield from 42.24% to 90.1%. A large excess of methyl acetate
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facilitates the shift in the forward direction resulting in an increase in the conversion of WCO
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to biodiesel. A further increase in the OMAMR to 1:15 resulted in a marginal decrease in the
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conversion of oil to biodiesel, which can be attributed to the fact that the usage of
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significantly higher molar ratio results in the excessive dilution giving enhanced reverse
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reaction [21]. The findings of the present investigation are in close agreement with the results
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of Du et al. [22] who reported optimum reactant molar ratio as 1:12 for the interesterification
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of crude soyabean oil. It was also reported that higher molar ratios beyond the optimum led to
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an excessive dilution of oil resulting in a reduced methyl esters yield.
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The effect of OMAMR on the biodiesel yield in the case of ultrasound-assisted enzymatic
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interesterification was also studied and the obtained results have been shown in Fig. 2(b). It
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can be seen from the Fig. 2(b) that, on increasing the OMAMR from 1:3 to 1:9, the methyl
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ester yield also proportionately increased. But a further increase in the OMAMR to 1:12 gave
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a marginal decrease in methyl ester yield establishing an optimum of 1:9 for the ultrasound
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assisted synthesis. It can be seen that the use of ultrasound assisted approach resulted in lower
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excess of methyl acetate as optimum and also higher yield as 96.1% was obtained in 3 h of
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reaction time. The obtained results can be attributed to the cavitational events produced due
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to the propagation of ultrasonic waves in liquid medium that can intensify the physical and
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chemical processing applications [23]. Considering the present work dealing with the
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biodiesel production, physical effects such as intense turbulence and liquid circulation
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currents (acoustic streaming) result in the formation of fine emulsion giving enhanced surface
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areas for reaction and enhanced rates of mass transfer, play a controlling role in deciding the
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intensification. Usai et al. [24] investigated interesterification reaction of methyl acetate with
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olive oil catalyzed by immobilized lipase from Candida antarctica, and reported a yield of
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80% at OMAMR of 1:20. Similarly, Xu et al. [25] reported a methyl ester yield of 67% for
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the interesterification reaction of refined soybean oil performed at atmospheric pressure,
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temperature of 40 °C, OMAMR of 1:12 for a reaction time of 36 h using Novozym enzyme.
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The main shortcoming of the enzymatic reactions as requirement of the long reaction time for
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high conversions [25] can be avoided with the use of ultrasound. The presented results in this
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work have established that ultrasound assisted approach reduces the excess reactant
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requirement and also reduces the reaction time considerably as compared to the conventional
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interesterification.
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3.2 Effect of enzyme loading
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The amount of enzyme used for the reaction is a critical aspect in deciding the cost of
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production and hence the applicability at the industrial scale applications. Thus due to the
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high cost of enzymes, it is important to optimize the quantity of enzyme used in the reaction 10
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mixture. The effect of enzyme loading on the biodiesel yield was investigated over the range
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of 2% to 8% (w/v) and the obtained results for the conventional approach have been shown in
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Fig. 3(a). It can be seen that, on increasing the enzyme loading from 2.0% to 6.0% (w/v), the
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methyl ester yield increased from 29.23 to 90.01%. However, when the loading was further
235
increased to 8.0% (w/v), the methyl ester yield was found to increase marginally to 91.2%.
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Evidently a high concentration of lipase shows plentiful activated sites and sufficient contact
237
giving higher yields. The optimum loading of the lipase enzyme for the methyl ester yield can
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be considered as 6% since there was no significant change in the methyl esters observed
239
beyond 6% (w/v) enzyme loading. The observed trend is in accordance with the results
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reported by Modi et al. [26], who reported an optimum loading of 10% for the Novozym-435
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enzyme for biodiesel production using interesterification reaction.
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One of the major objectives of the work was to develop a process that could require lower
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amount of enzyme and time to produce the maximum possible amount of biodiesel. Fig. 3(b)
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shows the effect of enzyme loading for the case of ultrasound assisted enzymatic
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interesterification. From Fig. 3(b), it is seen that an increase in the enzyme loading from 1.0%
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to 3.0% (w/v) resulted in significant increase in the yield of methyl esters. But further
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increase in the enzyme loading to 4.0% (w/v) did not result in significant increase in the
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biodiesel yield establishing 3% (w/v) enzyme loading as the optimum. It is also important to
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note that the obtained yield under optimum conditions for the ultrasound assisted approach is
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higher as compared to that obtained under the optimum conditions of enzyme loading
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(actually higher) for the conventional approach. The phenomenon of process intensification is
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very much evident from the results reported in Fig. 3(b). Ultrasonic irradiation significantly
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influences the enzyme catalyzed reactions by creating uniform mixing patterns or dispersions
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due to the physical effects of cavitation [27, 28]. Hence, in the case of ultrasound-assisted
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interesterification, to produce an equivalent amount of methyl esters, lower enzyme loading is 11
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required as compared to that required in the absence of ultrasound. Yu et al. [29] reported an
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optimum lipase enzyme (Novozym 435) loading as 6% to achieve 96% yield of methyl esters
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for ultrasound assisted enzymatic biodiesel production using ultrasonic bath. The required
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enzyme (Lipozyme TLIM) loading in the present work is 2 times lower as compared to the
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work of Yu et al. [29] using ultrasonic horn, which clearly establishes the significant process
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intensification aspects established in the present work.
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3.3 Effect of temperature
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Reaction temperature is an important parameter in deciding the progress of enzyme catalyzed
264
reactions and optimum temperature needs to be established. This is essential due to the fact
265
that higher temperature can help the substrate molecules to obtain adequate energy to
266
overcome the energy barrier and enhance the reaction rate, but significant increase in the
267
temperature can lead to the denaturation of enzyme. The effect of temperature has been
268
investigated over a temperature range from 30 to 50 °C for the conventional approach of
269
enzymatic interesterification. The obtained results have been presented in Fig. 4 where it can
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be seen that an increase in temperature from 30 to 40 °C gives a corresponding increase in the
271
biodiesel yield from 54.23 to 90.1%. The biodiesel yield reduced for a subsequent increase in
272
the reaction temperature from 40 to 50 °C. As the reaction temperature increases, the
273
collision frequency between enzyme and substrate molecules in the case of conventional
274
approach increases, which helps to form enzyme–substrate complexes at higher rates giving
275
increase in the biodiesel yield [29]. The maximum biodiesel yield was achieved at reaction
276
temperature of 40 ºC. The observed decrease in the biodiesel yield with further increase in the
277
temperature above 40 ºC is most likely because of the denaturation (alteration) of protein
278
structure originated from the heat-induced destruction of noncovalent interactions i.e. the
279
breakage of the weak ionic and hydrogen bonding that stabilizes the three dimensional
280
structure of the enzyme [30]. The optimum temperature of 40 ºC as obtained in the present 12
281
study is in agreement with the results reported by Sim et al. [31] for Thermomyces
282
lanuginosus lipase.
283
3.4 Effect of ultrasonic power
284
Ultrasonic power is an important factor influencing the degree of intensification of any
285
chemical processing application. The ultrasonic power decides the cavitational activity as
286
well as intensity and hence also would affect the biodiesel yield. The effect of ultrasonic
287
power has been investigated over the range of 40 W to 100 W and the obtained results have
288
been presented in Fig. 5. With an increase in ultrasonic power from 40 W to 80 W, a steady
289
increase in the biodiesel yield from 57.23% to 96.1% has been observed. For a further
290
increase in the power to 100 W, the biodiesel yield remained almost constant and hence 80 W
291
was taken as the optimum power dissipation. The introduction of ultrasound in the liquid
292
medium leads to the phenomenon of cavitation. With an increase in the power of ultrasonic
293
irradiation, the number of cavitation bubbles formed in the medium also increases giving
294
higher cavitational effects and hence the process intensification benefits [32]. The application
295
of ultrasound increases the interaction between enzyme and substrate, resulting in an
296
increased reaction rate. The energy generated due to cavitation is transferred to the reaction
297
system, which improves the process of mass transfer and diffusion of the substrate towards
298
the enzyme [33]. Because of these mechanisms, an increase in biodiesel yield was observed
299
till power of 80 W and the reaction time was also reduced from 24 h to 3 h due to the use of
300
ultrasonic irradiation for similar yields. The cavitational effects caused due to ultrasonic
301
irradiation are affected undesirably at higher power level. At very high power levels,
302
significant cavitation events can result in decoupling losses as well as cushioned cavity
303
collapse giving reduced energy transfer into the system and hence lower biodiesel yields. Ji et
304
al. [34] reported similar trends for biodiesel synthesis where ultrasonic power was varied
13
305
from 100 to 200 W at constant reactant ratio as 1:6 molar ratio in the presence of base
306
catalyst at 45 °C. The maximum biodiesel yield was obtained at the power of 150 W with
307
higher powers giving reduced biodiesel yields.. Another reason for marginal effect at higher
308
power dissipation can be the possible deactivation of the enzyme due to higher cavitational
309
intensity. It has been shown that the mild ultrasonic intensity can enhance the enzyme activity
310
but at higher ultrasonic power levels, the configuration of enzymes could be destroyed and
311
the enzymes could be denatured [35].
312
3.5 Effect of duty cycle
313
Ultrasonic duty cycle is another significant operating parameter deciding the economics of
314
the process as well as the yields. Duty cycle is the fraction of time during which an ultrasonic
315
system is operated. A duty cycle of 10%, for instance, is corresponding to ultrasonic
316
irradiation for 1 s followed by a rest period (without ultrasonic irradiation) of 9 s [36]. It is
317
also recommended to use ultrasonic irradiation with specific duty cycle to decrease the
318
possible deactivation of enzyme due to continuous ultrasonic irradiation. The effect of duty
319
cycle on biodiesel production was studied by varying ON–OFF time of ultrasonic irradiation.
320
The studies were performed using duty cycles in the range of 40% to 70% (higher duty cycles
321
were not used due to the wear problems and as per recommendations of the supplier). Effect
322
of duty cycle on biodiesel yield has been shown in Fig. 6 where it can be seen that the
323
biodiesel yield increases as the duty cycle increases from 40% (4 s ON and 6 s OFF) to 60%
324
(6 s ON and 4 s OFF) and thereafter no increase in the yield was observed for a further
325
increase in the duty cycle to 70% (7 s ON 3 s OFF). Using this optimum duty cycle of 60%,
326
maximum biodiesel yield of 96.1% was obtained in 3 h of reaction time. Irradiation of
327
ultrasound in continuous mode also can cause erosion of tip as well as lead to energy
328
intensive operation as compared to the pulsed mode. Pan et al. [37] have reported that
14
329
irradiation of ultrasound in both pulsed as well as continuous mode at a particular intensity
330
gave similar yield. Hence, it is established that proper ON–OFF duration of the specific
331
ultrasonic system for the desired application must be determined to get maximum product
332
yield, as unnecessary increase in the ON time would result in the excessive heating and
333
unnecessary electrical energy consumption.
334
3.6. Reusability of enzyme
335
There is a necessity to reuse the biocatalyst after every cycle as enzyme plays a very vital part
336
in the commercial profitability of enzyme catalyzed reactions. Therefore, reusability study
337
was critically vital for the economic feasibility of reaction. Reusability study was carried out
338
using optimized parameters. After each cycle, the immobilized lipase enzyme was filtered off
339
followed by washing with acetone and then dried in oven for 30 min at 40 °C and then kept in
340
the desiccator at room temperature (30 ± 2 °C). The recycled enzyme was used for the next
341
cycle. The results obtained after seven cycles are depicted in Fig. 7(a) which demonstrates
342
that the biodiesel yield generally decreases with an increase in the number of cycles. Also it
343
has been observed that the catalytic activity of immobilized lipase also decreased after each
344
cycle as shown in Fig. 7(b). Only 25% of original enzyme activity was retained after 7 cycles.
345
The obtained results are in well agreement with the Zhang et al. [38] where only 20%
346
conversion was achieved after 6 cycles of reuse for the Lipozyme TLIM enzyme without
347
ultrasonic irradiation. Paludo et al. [39] have also reported that enzyme activity of Lipozyme
348
TLIM decreases completely in only 4 cycles in the presence of mechanical agitation. The
349
decrease in activity and decrease in biodiesel yield may be attributed to the denaturation and
350
breakage of bonding between enzyme and supporting media upon use [40]. Thus it can be
351
established that Lipozyme TLIM activity decreases after repeated use in the biodioesel
352
synthesis reaction. Based on the comparison of the data available in the literature for the
15
353
conventional approach, it is expected that use of ultrasound under the set of optimum
354
conditions does not contribute significantly to enhance the deactivation, especially in the first
355
few cycles of treatment.
356
3.7. Structural characterisation of immobilised lipase
357
The influence of ultrasonic irradiation on the secondary structure of enzyme was analysed by
358
CD spectroscopy. The structural change of the lipase was compared to the ultrasound
359
irradiated enzyme (after use in the multiple cycles) through CD spectra analysis as shown in
360
Fig. 8. Intensity of CD peaks indicates the extent of ellipticity in the protein [41], which
361
shows the changes in protein structure. Ultrasound-treated Lipozyme TLIM showed
362
significant changes in the CD spectra, which reflected the structural changes in protein
363
secondary structure. Compared to non-sonicated enzyme, the amount of α-helix in ultrasound
364
irradiated-immobilized enzyme decreased, whereas β-sheet and unordered polypeptide
365
amount increased as shown in Table 2. Ultrasound irradiated immobilised lipase retained
366
about 79% of its native α- helix content, as obtained from the mean residue ellipticity at 222
367
nm. Application of ultrasound resulted in pressure-induced spectral changes which were
368
indicative of change in the proportion of α-helical and β-sheet structure in the protein. The
369
secondary structure of protein not only depends on the amino acid sequence but also on the
370
interactions between different parts of the molecule [42]. Stathpulos et al. [43] have reported
371
similar findings for the changes in the enzyme structure.
372
3.8. Surface morphology of immobilized lipase
373
The SEM images of the untreated and ultrasound irradiated immobilized lipase samples
374
shown in Fig. 9 revealed that morphological changes were brought about by ultrasonication.
375
The surface of unused enzyme was more or less smooth, while after irradiation, the structure
376
was open though small holes were observed on the surface, which can be attributed to the 16
377
indentations introduced by the cavitational effects. Comparison of SEM images of the
378
immobilized enzyme before and after 7 cycles of use in ultrasound-assisted reaction shows
379
that there is a substantial change in morphology. Thus, ultrasonic treatment caused alterations
380
in Lipozyme TLIM both in terms of the morphology as well as structure at the molecular
381
level. It is important to note that some degree of morphological changes are expected to
382
increase the activity due to the enhanced access of the substrate molecules to the active
383
catalytic sites. Still continuous use, especially under conditions of intense cavitation, would
384
be detrimental contributing to the decrease in the activity of the enzyme. Zhang et al. [42]
385
also reported similar observation for ultrasound irradiated Candida Antarctica lipase B
386
(CalB), where surface was quite different from the initial morphology of lipase without
387
sonication.
388
4. Conclusions
389
Novel
390
interesterification of WCO with methyl acetate has been investigated. Methyl acetate proved
391
to be efficient acyl acceptor giving significant yields of biodiesel. Thus the approach
392
presented in the work avoids the use of methanol and hence possible deactivation of enzyme
393
due to the use of methanol could be avoided. Ultrasound-assisted approach resulted in
394
significant intensification in the process with reduction in reaction from 24 h to 3 h as
395
compared to conventional stirring method. Low frequency mild ultrasonic irradiation was
396
established to be quite important to enhance the rate of enzymatic reaction. The optimum
397
ultrasound parameters observed were 20 kHz frequency, 80 W power with 60% duty cycle.
398
Use of ultrasound also allowed working with lower enzyme loading as compared with
399
conventional approach for giving similar biodiesel yields which also should reduce the cost
400
of production. Overall, it has been established that the interesterification of WCO with
approach
for
ultrasound-assisted
enzyme
(Lipozyme
TLIM)
catalyzed
17
401
methyl acetate using Lipozyme TLIM under ultrasonic irradiation gives an interesting
402
alternative for synthesis of biodiesel with significant process intensification benefits.
403
404
Acknowledgment
405
Authors acknowledge the funding of Department of Science and Technology and Portuguese
406
Science Foundation under the Indo-Portuguese collaborative program (Project Reference:
407
DST/INT/PORTUGAL/P-11/2013)
408
409
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410
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402–407.
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vibration for biodiesel production from soybean oil by Novozym 435, Process Biochem.
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45 (2010) 519–525.
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of forward and reverse reactions, Enzyme Microb. Technol. 38 (2006) 814–820.
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and Stability of Immobilized Thermomyces lanuginosus in Producing Fatty Acid Methyl
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Ester (FAME), IJSRP 3 ( 2013) 1–4.
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[32] P. Eisenberg, Cavitation, Handbook of Fluid Mechanics, Tata McGraw Hill Publications, 1984 (Chapter-12).
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[37] Z. Pan, W. Qu, H. Ma, G.G. Atungulu, T.H. McHugh, Continuous and pulsed ultrasound
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(2011) 1249–1257.
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by immobilized Thermomyces lanuginosus lipase (Lipozyme TLIM), Bioprocess
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Biosyst. Eng. 34 (2011) 1163–1168.
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[39] N. Paludo, J.S. Alves, C. Altmann, M.A.Z. Ayub, R. Fernandez-Lafuente, R.C.
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Rodrigues, The combined use of ultrasound and molecular sieves improves the synthesis
511
of ethyl butyrate catalyzed by immobilized Thermomyces lanuginosus lipase, Ultrason.
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Sonochem. 22 (2015) 89–94.
513
[40] G.V. Waghmare, M.D. Vetal, V.K. Rathod, Ultrasound assisted enzyme catalyzed
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synthesis of glycerol carbonate from glycerol and dimethyl carbonate, Ultrason.
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Sonochem. 22 (2015) 311–316.
22
516
[41] N. Sreerama, S. Venyaminov, R.W. Woody, Estimation of protein secondary structure
517
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518
the analysis, Anal. Biochem. 287 (2000) 243–251.
519
[42] J.C. Zhang, C. Zhang, L. Zhao, C.T. Wang, Lipase-catalyzed synthesis of sucrose fatty
520
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521
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522
[43] P.B. Stathopulos, G.A. Scholz, Y.M. Hwang, J.A. Rumfeldt, J.R. Lepock, E.M.
523
Meiering, Sonication of proteins causes formation of aggregates that resemble amyloid,
524
Protein Sci. 13 (2004) 3017‒3027.
525
526
527
23
528
List of Figures
529
Scheme 1. Production of fatty acid methyl esters by enzymatic interesterification of
530
triglyceride with methyl acetate
531 532
Fig. 1. Experimental set up of ultrasound-assisted interesterification reaction
533
Fig. 2(a). Effect of molar ratio on the biodiesel yield for the conventional approach of
534
enzymatic biodiesel production
535
Fig. 2(b). Effect of molar ratio on the biodiesel yield for the ultrasound-assisted approach of
536
enzymatic biodiesel production
537
Fig. 3(a). Effect of enzyme loading on the biodiesel yield for the conventional approach of
538
biodiesel production
539
Fig. 3(b). Effect of enzyme loading on the biodiesel yield for the ultrasound-assisted
540
approach of biodiesel production
541
Fig. 4. Effect of temperature on enzymatic biodiesel yield
542
Fig. 5. Effect of ultrasonic power on the biodiesel yield for ultrasound-assisted approach of
543
enzymatic biodiesel production
544
Fig. 6. Effect of duty cycle on the biodiesel yield for ultrasound-assisted approach of
545
enzymatic biodiesel production
546
Fig. 7(a). Effect of reusability of enzyme on the biodiesel yield for ultrasound-assisted
547
approach of enzymatic biodiesel production
548
Fig. 7(b). Effect of reusability on the relative enzyme activity for ultrasound-assisted
549
approach of enzymatic biodiesel production
550
Fig. 8. CD spectra of unused and reused immobilized lipase
551
Fig. 9. SEM images of: (A) Unused immobilized lipase and (B): Reused immobilized lipase
552
24
553
List of Tables
554
Table 1. Composition and properties of waste cooking oil
555
Table 2. Contents of secondary structure of non-sonicated and sonicated immobilized lipase
556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572
25
573 574 575
Scheme 1: Production of fatty acid methyl esters by enzymatic interesterification of
576
triglyceride with methyl acetate
577
Generator
Horn
Reactor
Water out Reaction mixture Drain
Water in
578 579
Fig. 1. Experimental set up of ultrasound-assisted interesterification reaction
580
26
581 582
Fig. 2(a). Effect of molar ratio on the biodiesel yield for the conventional approach of
583
enzymatic biodiesel production
584 585
Fig. 2(b). Effect of molar ratio on the biodiesel yield for the ultrasound-assisted approach of
586
enzymatic biodiesel production
27
587 588
Fig. 3(a). Effect of enzyme loading on the biodiesel yield for the conventional approach of
589
biodiesel production
590 591
Fig. 3(b). Effect of enzyme loading on the biodiesel yield for the ultrasound-assisted
592
approach of biodiesel production
28
593 594
Fig. 4. Effect of temperature on the biodiesel yield for the conventional approach of
595
enzymatic biodiesel production
596 597
Fig. 5. Effect of ultrasonic power on the biodiesel yield for ultrasound-assisted approach of
598
enzymatic biodiesel production
29
599 600
Fig. 6. Effect of duty cycle on the biodiesel yield for ultrasound-assisted approach of
601
enzymatic biodiesel production
602 603
Fig. 7(a). Effect of reusability of enzyme on the biodiesel yield for ultrasound-assisted
604
approach of enzymatic biodiesel production
605 30
606 607
Fig. 7(b). Effect of reusability on the relative enzyme activity for ultrasound-assisted
608
approach of enzymatic biodiesel production
609
610 611
Fig. 8. CD spectra of unused and reused immobilized lipase
31
(A) Unused immobilized lipase
(B) Reused immobilized lipase
612 613
Fig. 9. SEM images of: (A) Unused immobilized lipase and (B): Reused immobilized lipase
614 615 616 617
32
618
Table 1. Composition and properties of waste cooking oil Property
Value
Linoleic acid (%)
73.4
Oleic acid (%)
18.3
Palmitic acid (%)
6.7
Stearic acid (%)
1.6
Saponification value (mg KOH/g of oil)
198
Density (kg/m3)
930
Acid value (mg KOH/g oil)
4.3
Viscosity (mm2/s)
54.3
619 620
Table 2. Contents of secondary structure of non-sonicated and sonicated immobilized lipase α-Helix
β-Sheet
β-Turn
Random
Relative enzyme
(%)
(%)
(%)
coil (%)
activity (%)
Non-sonicated
52.51
15.43
11.92
18.42
100
Sonicated
41.61
13.62
19.96
22.98
25.1
Treatment
621 622
33
623
Research Highlights
624 625
•
Intensified enzymatic biodiesel production using interesterification approach
626
•
Detailed investigation into the effect of different operating parameters.
627
•
Ultrasonic method gave significant benefits than conventional stirring method.
628
•
Significant reduction in time and requirement of lower loading
629
630 631 632 633 634 635
34
S1350-4177(15)30027-4 http://dx.doi.org/10.1016/j.ultsonch.2015.09.006 ULTSON 2990
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
30 June 2015 5 September 2015 6 September 2015
Please cite this article as: P.B. Subhedar, P.R. Gogate, Ultrasound assisted intensification of biodiesel production using enzymatic interesterification, Ultrasonics Sonochemistry (2015), doi: http://dx.doi.org/10.1016/j.ultsonch. 2015.09.006
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1
Ultrasound assisted intensification of biodiesel production using enzymatic
2
interesterification
3
4
Preeti B. Subhedar, Parag R. Gogate*
5
6
7
8
Chemical Engineering Department,
9
Institute of Chemical Technology,
10
Matunga, Mumbai – 400 019, India
11
12
13
14
15
*
16
Tel.: +91 22 33612024,
17
Fax: +91 22 33611020;
18
Corresponding author
E-mail address: [email protected]
1
19
Abstract
20
Ultrasound assisted intensification of synthesis of biodiesel from waste cooking oil
21
using methyl acetate and immobilized lipase obtained from Thermomyces lanuginosus
22
(Lipozyme TLIM) as a catalyst has been investigated in the present work. The reaction has
23
also been investigated using the conventional approach based on stirring so as to establish the
24
beneficial effects obtained due to the use of ultrasound. Effect of operating conditions such as
25
reactant molar ratio (oil and methyl acetate), temperature and enzyme loading on the yield of
26
biodiesel has been investigated. Optimum conditions for the conventional approach (without
27
ultrasound) were established as reactant molar ratio of 1:12 (oil : methyl acetate), enzyme
28
loading of 6% (w/v), temperature of 40 ºC and reaction time of 24 h and under these
29
conditions, 90.1% biodiesel yield was obtained. The optimum conditions for the ultrasound
30
assisted approach were oil to methyl acetate molar ratio of 1:9, enzyme loading of 3% (w/v),
31
and reaction time of 3 h and the biodiesel yield obtained under these conditions was 96.1%.
32
Use of ultrasound resulted in significant reduction in the reaction time with higher yields and
33
lower requirement of the enzyme loading. The obtained results have clearly established that
34
ultrasound assisted interesterification was a fast and efficient approach for biodiesel
35
production giving significant benefits, which can help in reducing the costs of production.
36
Reusability studies for the enzyme were also performed but it was observed that reuse of the
37
catalyst under the optimum experimental condition resulted in reduced enzyme activity and
38
biodiesel yield.
39
40
Key words: Ultrasound, Intensification, Interesterification, Biodiesel, Immobilized Lipase,
41
Operating parameters
2
42
1. Introduction
43
Biodiesel offers a substitute to the petroleum based fuels offering greener processing and
44
reduced emissions. Biodiesel (monoalkyl fatty acid methyl esters), which is generally
45
produced by the transesterification reaction of triglycerides with methanol, has become
46
significantly important in recent years due to the diminishing petroleum reserves, strong
47
dependence of the national economies on the fuel prices and the ecological concerns of
48
released gases from petroleum-based fuel [1]. Even though biodiesel has been generally
49
produced using the chemical synthesis approach, there are numerous problems associated
50
with this approach such as recovery of glycerol, removal of inorganic salts and significant
51
processing costs especially with the separation of excess methanol being used in the synthesis
52
[2, 3].
53
Enzyme catalyzed approach for production of biodiesel partially addresses these issues and
54
offers an environmentally more attractive alternative to the conventional processes [4-6]. The
55
enzyme catalyzed biodiesel production under moderate environments allows the easy
56
removal of the biocatalyst and glycerol recovery by centrifugation resulting in achieving
57
biodiesel production with high purity in simple steps [7]. Typically short-chain alcohols such
58
as methanol are used as the acyl acceptor for biodiesel production, however, usage of
59
methanol in excess leads to deactivation of enzyme, and glycerol which is a major by-product
60
of the reaction, also has inhibitory effects on the enzymatic activity apart from offering low
61
commercial value. These associated issues with the enzymatic route have offered
62
considerable restrictions for the effective application at industrial scale [8]. Replacing the
63
alcohol as alkyl acceptor by alkyl acetate helps to solve these problems due to the use of
64
alcohol and the reaction is described as interesterification.
3
65
The present work deals with production of biodiesel using the interesterification reaction
66
based on methyl acetate as an acyl acceptor instead of methanol and the by-product formed in
67
the reaction is triacetin instead of glycerol (Scheme 1). Triacetin has higher commercial value
68
as compared to glycerol due to the use as a gelatinizing agent as well as additives in tobacco,
69
pharmaceutical and cosmetic industries [9]. Moreover, triacetin has no adverse effect on the
70
activity of lipase enzyme [10]. Though interesterification offers significant advantages, very
71
few papers associated to this reaction route have been reported. Kim et al. [11] used ethyl
72
acetate as acyl acceptor and highest biodiesel production yield of 63.3% was achieved by
73
using an ethyl acetate to oil molar ratio of 6:1 molar ratio with 8% of the immobilized lipase
74
Novozym 435.
75
synthesis of biodiesel based on the use of methyl acetate instead of more commonly used
76
alcohol. Maximum biodiesel yield of 92.34% was obtained by using 1.5 g of enzyme bead,
77
1:12 molar ratio of oil to methyl acetate, temperature of 35 °C in 60 h of reaction time.
78
Apart from possible inactivation of enzyme, another key shortcoming in the
79
commercialization of biodiesel production is the cost of operation especially dominated by
80
the higher costs of the enzyme, raw materials and significantly longer reaction times. Even
81
though, biodiesel has an enormous potential to substitute exhaustible fossil fuel, for all the
82
current processing technologies, the cost of biodiesel is about 50 to 100% higher than the
83
petroleum based diesel fuel [13]. The significant cost associated with the raw materials [14]
84
can be reduced by using waste cooking oil (WCO) or other sustainable resources. The mass
85
transfer limitations due to the heterogeneous nature of reactants can be reduced based on the
86
use of intensification approaches such as using ultrasound. Use of interesterification approach
87
instead of transesterification can also help in producing a more valuable co-product with the
88
biodiesel. Considering these aspects, interesterification of waste cooking oil using enzymatic
89
route and intensification using ultrasound has been investigated in the present work.
Surendhiran and Vijay [12] studied the interesterification reaction for
4
90
Application of ultrasound can give substantial degree of process intensification based on
91
cavitation phenomenon. The cavitational effects can improve the mass transfer at mild
92
reaction conditions in terms of temperature and pressure resulting into faster reaction rate,
93
higher product yield [15, 16] and possibly requirement of lower acyl acceptor to oil molar
94
ratio and catalyst loading. Cavitation creates intense turbulence and liquid circulation at
95
micro scale which help in reducing the mass transfer resistances in heterogeneous systems
96
[17]. Although researchers have used ultrasound for the enhancement of transesterification
97
route for biodiesel synthesis [18-20], there is practically no information available for its
98
application for the enzymatic interesterification route of biodiesel production. The main aim
99
of the present work was to investigate the influence of ultrasound on the enzymatic
100
interesterification of waste cooking oil using immobilized lipase. Furthermore, different
101
reaction conditions including molar ratio of substrates, enzyme loading, reaction temperature
102
and time which might influence the yield of FAMEs have been investigated so as to
103
maximize the yield. Comparison of the ultrasound based approach with the conventional
104
approach has been presented so as to establish the benefits of using ultrasound as a source of
105
mixing.
106
107
2. Materials and methods
108
2.1. Materials
109
Waste cooking oil (WCO) was obtained from a local restaurant in Mumbai, India. The initial
110
analysis of oil revealed that the main contents were unsaturated fatty acids (linoleic and oleic
111
acids as a total of 91%) with very less quantum of saturated fatty acids (palmitic and stearic
112
acid). The properties of WCO used in the work for the biodiesel production are shown in
113
Table 1. Methyl acetate was acquired from S.D. Fine Chemicals Ltd., Mumbai. Acetonitrile 5
114
and acetone (HPLC grade) used as HPLC solvents were obtained from J.T. Baker, Mumbai.
115
The methyl oleate and methyl linoleate standards were procured from Sigma-Aldrich. Lipase
116
enzyme, Lipozyme TL IM, immobilized on silica granules, was kindly provided as a gift
117
sample by Brenntag India Pvt. Ltd., Mumbai, India.
118
2.2.2. Interesterification of waste cooking oil
119
A 100 mL glass reactor equipped with mechanical stirrer and baffles to avoid vortex
120
formation was used for the conventional approach of interesterification. The temperature of
121
the reaction was maintained constant using water bath. Reactor was also equipped with a
122
condenser to achieve complete reflux conditions and recycle the methyl acetate vapours back
123
to the reaction mixture. WCO and methyl acetate were first fed to the reactor and heating was
124
started. After reaching the desired temperature, lipase enzyme was added into the reactor. 0.5
125
ml samples were withdrawn at specific intervals and analysed using HPLC. It was observed
126
that the pH of the reaction mixture did not change with time. The effect of operating
127
parameters such as reactant molar ratio, temperature and enzyme loading has also been
128
investigated. The reproducibility of the data was tested by performing the experiments in
129
duplicate and average values have been reported. The observed errors were within 2% of the
130
reported average value.
131
2.2.3. Ultrasound assisted interesterification of waste cooking oil
132
Ultrasonic horn obtained from Dakshin, Mumbai was used for the approach of ultrasound-
133
assisted enzymatic hydrolysis. The ultrasonic irradiation at a frequency of 20 kHz was
134
transferred into the reaction mixture through a titanium cylindrical horn, submerged 2.0 cm
135
into reaction mixture. Ultrasonic horn had a diameter of 1.1 cm and maximum rated power
136
output of 120 W. The schematic representation of experimental set up has been shown in Fig.
137
1. The procedure followed for the ultrasound-assisted enzymatic interesterification was 6
138
similar to that mentioned in the conventional approach of enzymatic interesterification but the
139
samples were withdrawn after 30 min for methyl ester analysis. The temperature and flow
140
rate of circulating water bath was maintained in such a way that the reaction temperature was
141
always controlled to 50 °C. The effect of different parameters such as ultrasonic power,
142
ultrasonic duty cycle, oil to methyl acetate molar ratio and enzyme loading has been
143
investigated.
144
2.3. Analytical methods
145
2.3.1. Acid value
146
The acid value of the waste cooking oil was determined by the acid-base titration technique
147
as per the details mentioned in our earlier work [9].
148
2.3.2. Fatty acid composition and fatty acid methyl ester analysis
149
The original composition of the fatty acids in the oil were determined by converting all the
150
fatty acids into corresponding esters and subsequently analyzed using gas chromatography
151
(GC) based on the use of BP-X70 column. The progress of the reaction was monitored in
152
terms of the fatty acid methyl ester content using Agilent Eclipse XDB C-18 HPLC column,
153
having dimensions of 4.6 × 250 mm. Acetonitrile: acetone (70:30 v/v ratio) was used as a
154
mobile phase and samples were analyzed isocratically at a flow rate of 1.5 mL/min. Samples
155
for analysis were prepared by diluting 10µL of the reaction mixture in 10 mL of mobile
156
phase. The concentration was determined based on the method of calculating the area under
157
the peak obtained at the specific retention time.
158
2.3.4. Enzyme assay
159
The enzyme assay was done using tributyrin as a substrate for enzyme. Tributyrin (0.2 mL)
160
was incubated with the lipase enzyme (200 mg) in phosphate buffer (pH 7) for 5 min. After 5
7
161
min of incubation, the reaction was ended by the addition of methanol (20 mL) and reaction
162
mixture was titrated against alcoholic NaOH (0.1 M) using phenolphthalein as an indicator.
163
Immobilized enzyme activity can be expressed as µmoles of ester formed per minute and was
164
calculated using the following equation:
165
Enzyme activity (TBU/g) =
166
where V = volume in ml of NaOH which is a measure of tributyrin consumed during
167
reaction, M = molarity of NaOH, E = amount of enzyme employed in mg and T = time of
168
reaction in min. One unit of lipase activity was defined as the amount of enzyme required to
169
hydrolyze 1 mole of ester bond per minute under assay conditions.
170
2.3.4. Structural characterisation of immobilised lipase
171
Circular dichroism (CD) spectroscopy was used to monitor the secondary structure of the
172
non-sonicated and ultrasound irradiated immobilised lipase enzyme. The CD spectra were
173
recorded on a JASCO J-810 CD instrument (JASCO). CD spectra were scanned in the far UV
174
range (200 to 250 nm) with three replicates at 50 nm/min with bandwidth of 0.5 nm. Cell
175
length was 10 mm. In all measurements, the lipase concentration was kept constant at 50
176
mg/mL. The CD data were expressed in terms of ellipticity in mdeg. All the spectra were
177
corrected by subtracting a blank spectrum (without lipase).
178
2.3.5. Environmental scanning electron microscopy (ESEM)
179
To understand the effect of ultrasonic irradiation on the immobilized lipase, samples were
180
analyzed by environmental scanning electron microscopy. The analysis of the enzyme was
181
performed before and after the ultrasonic irradiation. Samples were analyzed by Quanta 200
182
unit (FEI, Field Emission Instruments, Hillsboro, OR) at 20 kV for determination of the
V × M × 1000 W× T
8
183
morphological changes occurred due to the use of ultrasound. Samples of immobilized
184
lipases were displaced in metallic plates and submitted to a previous treatment of atomization
185
with gold to improve the image definition and characteristics.
186
187
3. Results and discussion
188
3.1 Effect of reactant molar ratio
189
In the interesterification reaction, one mole of triglycerides reacts with three moles of methyl
190
acetate giving one mole of biodiesel, indicating a stochiometric requirement of oil to methyl
191
acetate molar ratio (OMAMR) as 1:3. An excess of methyl acetate is typically used to drive
192
the reaction in the forward direction. Experiments were performed with OMAMR ranging
193
from 1:6 to 1:15. Fig. 2(a) depicts the obtained results for the conventional approach of
194
stirring, where it can be seen that an increase in the molar ratio from 1:6 to 1:12 results in an
195
increase in the biodiesel yield from 42.24% to 90.1%. A large excess of methyl acetate
196
facilitates the shift in the forward direction resulting in an increase in the conversion of WCO
197
to biodiesel. A further increase in the OMAMR to 1:15 resulted in a marginal decrease in the
198
conversion of oil to biodiesel, which can be attributed to the fact that the usage of
199
significantly higher molar ratio results in the excessive dilution giving enhanced reverse
200
reaction [21]. The findings of the present investigation are in close agreement with the results
201
of Du et al. [22] who reported optimum reactant molar ratio as 1:12 for the interesterification
202
of crude soyabean oil. It was also reported that higher molar ratios beyond the optimum led to
203
an excessive dilution of oil resulting in a reduced methyl esters yield.
204
The effect of OMAMR on the biodiesel yield in the case of ultrasound-assisted enzymatic
205
interesterification was also studied and the obtained results have been shown in Fig. 2(b). It
9
206
can be seen from the Fig. 2(b) that, on increasing the OMAMR from 1:3 to 1:9, the methyl
207
ester yield also proportionately increased. But a further increase in the OMAMR to 1:12 gave
208
a marginal decrease in methyl ester yield establishing an optimum of 1:9 for the ultrasound
209
assisted synthesis. It can be seen that the use of ultrasound assisted approach resulted in lower
210
excess of methyl acetate as optimum and also higher yield as 96.1% was obtained in 3 h of
211
reaction time. The obtained results can be attributed to the cavitational events produced due
212
to the propagation of ultrasonic waves in liquid medium that can intensify the physical and
213
chemical processing applications [23]. Considering the present work dealing with the
214
biodiesel production, physical effects such as intense turbulence and liquid circulation
215
currents (acoustic streaming) result in the formation of fine emulsion giving enhanced surface
216
areas for reaction and enhanced rates of mass transfer, play a controlling role in deciding the
217
intensification. Usai et al. [24] investigated interesterification reaction of methyl acetate with
218
olive oil catalyzed by immobilized lipase from Candida antarctica, and reported a yield of
219
80% at OMAMR of 1:20. Similarly, Xu et al. [25] reported a methyl ester yield of 67% for
220
the interesterification reaction of refined soybean oil performed at atmospheric pressure,
221
temperature of 40 °C, OMAMR of 1:12 for a reaction time of 36 h using Novozym enzyme.
222
The main shortcoming of the enzymatic reactions as requirement of the long reaction time for
223
high conversions [25] can be avoided with the use of ultrasound. The presented results in this
224
work have established that ultrasound assisted approach reduces the excess reactant
225
requirement and also reduces the reaction time considerably as compared to the conventional
226
interesterification.
227
3.2 Effect of enzyme loading
228
The amount of enzyme used for the reaction is a critical aspect in deciding the cost of
229
production and hence the applicability at the industrial scale applications. Thus due to the
230
high cost of enzymes, it is important to optimize the quantity of enzyme used in the reaction 10
231
mixture. The effect of enzyme loading on the biodiesel yield was investigated over the range
232
of 2% to 8% (w/v) and the obtained results for the conventional approach have been shown in
233
Fig. 3(a). It can be seen that, on increasing the enzyme loading from 2.0% to 6.0% (w/v), the
234
methyl ester yield increased from 29.23 to 90.01%. However, when the loading was further
235
increased to 8.0% (w/v), the methyl ester yield was found to increase marginally to 91.2%.
236
Evidently a high concentration of lipase shows plentiful activated sites and sufficient contact
237
giving higher yields. The optimum loading of the lipase enzyme for the methyl ester yield can
238
be considered as 6% since there was no significant change in the methyl esters observed
239
beyond 6% (w/v) enzyme loading. The observed trend is in accordance with the results
240
reported by Modi et al. [26], who reported an optimum loading of 10% for the Novozym-435
241
enzyme for biodiesel production using interesterification reaction.
242
One of the major objectives of the work was to develop a process that could require lower
243
amount of enzyme and time to produce the maximum possible amount of biodiesel. Fig. 3(b)
244
shows the effect of enzyme loading for the case of ultrasound assisted enzymatic
245
interesterification. From Fig. 3(b), it is seen that an increase in the enzyme loading from 1.0%
246
to 3.0% (w/v) resulted in significant increase in the yield of methyl esters. But further
247
increase in the enzyme loading to 4.0% (w/v) did not result in significant increase in the
248
biodiesel yield establishing 3% (w/v) enzyme loading as the optimum. It is also important to
249
note that the obtained yield under optimum conditions for the ultrasound assisted approach is
250
higher as compared to that obtained under the optimum conditions of enzyme loading
251
(actually higher) for the conventional approach. The phenomenon of process intensification is
252
very much evident from the results reported in Fig. 3(b). Ultrasonic irradiation significantly
253
influences the enzyme catalyzed reactions by creating uniform mixing patterns or dispersions
254
due to the physical effects of cavitation [27, 28]. Hence, in the case of ultrasound-assisted
255
interesterification, to produce an equivalent amount of methyl esters, lower enzyme loading is 11
256
required as compared to that required in the absence of ultrasound. Yu et al. [29] reported an
257
optimum lipase enzyme (Novozym 435) loading as 6% to achieve 96% yield of methyl esters
258
for ultrasound assisted enzymatic biodiesel production using ultrasonic bath. The required
259
enzyme (Lipozyme TLIM) loading in the present work is 2 times lower as compared to the
260
work of Yu et al. [29] using ultrasonic horn, which clearly establishes the significant process
261
intensification aspects established in the present work.
262
3.3 Effect of temperature
263
Reaction temperature is an important parameter in deciding the progress of enzyme catalyzed
264
reactions and optimum temperature needs to be established. This is essential due to the fact
265
that higher temperature can help the substrate molecules to obtain adequate energy to
266
overcome the energy barrier and enhance the reaction rate, but significant increase in the
267
temperature can lead to the denaturation of enzyme. The effect of temperature has been
268
investigated over a temperature range from 30 to 50 °C for the conventional approach of
269
enzymatic interesterification. The obtained results have been presented in Fig. 4 where it can
270
be seen that an increase in temperature from 30 to 40 °C gives a corresponding increase in the
271
biodiesel yield from 54.23 to 90.1%. The biodiesel yield reduced for a subsequent increase in
272
the reaction temperature from 40 to 50 °C. As the reaction temperature increases, the
273
collision frequency between enzyme and substrate molecules in the case of conventional
274
approach increases, which helps to form enzyme–substrate complexes at higher rates giving
275
increase in the biodiesel yield [29]. The maximum biodiesel yield was achieved at reaction
276
temperature of 40 ºC. The observed decrease in the biodiesel yield with further increase in the
277
temperature above 40 ºC is most likely because of the denaturation (alteration) of protein
278
structure originated from the heat-induced destruction of noncovalent interactions i.e. the
279
breakage of the weak ionic and hydrogen bonding that stabilizes the three dimensional
280
structure of the enzyme [30]. The optimum temperature of 40 ºC as obtained in the present 12
281
study is in agreement with the results reported by Sim et al. [31] for Thermomyces
282
lanuginosus lipase.
283
3.4 Effect of ultrasonic power
284
Ultrasonic power is an important factor influencing the degree of intensification of any
285
chemical processing application. The ultrasonic power decides the cavitational activity as
286
well as intensity and hence also would affect the biodiesel yield. The effect of ultrasonic
287
power has been investigated over the range of 40 W to 100 W and the obtained results have
288
been presented in Fig. 5. With an increase in ultrasonic power from 40 W to 80 W, a steady
289
increase in the biodiesel yield from 57.23% to 96.1% has been observed. For a further
290
increase in the power to 100 W, the biodiesel yield remained almost constant and hence 80 W
291
was taken as the optimum power dissipation. The introduction of ultrasound in the liquid
292
medium leads to the phenomenon of cavitation. With an increase in the power of ultrasonic
293
irradiation, the number of cavitation bubbles formed in the medium also increases giving
294
higher cavitational effects and hence the process intensification benefits [32]. The application
295
of ultrasound increases the interaction between enzyme and substrate, resulting in an
296
increased reaction rate. The energy generated due to cavitation is transferred to the reaction
297
system, which improves the process of mass transfer and diffusion of the substrate towards
298
the enzyme [33]. Because of these mechanisms, an increase in biodiesel yield was observed
299
till power of 80 W and the reaction time was also reduced from 24 h to 3 h due to the use of
300
ultrasonic irradiation for similar yields. The cavitational effects caused due to ultrasonic
301
irradiation are affected undesirably at higher power level. At very high power levels,
302
significant cavitation events can result in decoupling losses as well as cushioned cavity
303
collapse giving reduced energy transfer into the system and hence lower biodiesel yields. Ji et
304
al. [34] reported similar trends for biodiesel synthesis where ultrasonic power was varied
13
305
from 100 to 200 W at constant reactant ratio as 1:6 molar ratio in the presence of base
306
catalyst at 45 °C. The maximum biodiesel yield was obtained at the power of 150 W with
307
higher powers giving reduced biodiesel yields.. Another reason for marginal effect at higher
308
power dissipation can be the possible deactivation of the enzyme due to higher cavitational
309
intensity. It has been shown that the mild ultrasonic intensity can enhance the enzyme activity
310
but at higher ultrasonic power levels, the configuration of enzymes could be destroyed and
311
the enzymes could be denatured [35].
312
3.5 Effect of duty cycle
313
Ultrasonic duty cycle is another significant operating parameter deciding the economics of
314
the process as well as the yields. Duty cycle is the fraction of time during which an ultrasonic
315
system is operated. A duty cycle of 10%, for instance, is corresponding to ultrasonic
316
irradiation for 1 s followed by a rest period (without ultrasonic irradiation) of 9 s [36]. It is
317
also recommended to use ultrasonic irradiation with specific duty cycle to decrease the
318
possible deactivation of enzyme due to continuous ultrasonic irradiation. The effect of duty
319
cycle on biodiesel production was studied by varying ON–OFF time of ultrasonic irradiation.
320
The studies were performed using duty cycles in the range of 40% to 70% (higher duty cycles
321
were not used due to the wear problems and as per recommendations of the supplier). Effect
322
of duty cycle on biodiesel yield has been shown in Fig. 6 where it can be seen that the
323
biodiesel yield increases as the duty cycle increases from 40% (4 s ON and 6 s OFF) to 60%
324
(6 s ON and 4 s OFF) and thereafter no increase in the yield was observed for a further
325
increase in the duty cycle to 70% (7 s ON 3 s OFF). Using this optimum duty cycle of 60%,
326
maximum biodiesel yield of 96.1% was obtained in 3 h of reaction time. Irradiation of
327
ultrasound in continuous mode also can cause erosion of tip as well as lead to energy
328
intensive operation as compared to the pulsed mode. Pan et al. [37] have reported that
14
329
irradiation of ultrasound in both pulsed as well as continuous mode at a particular intensity
330
gave similar yield. Hence, it is established that proper ON–OFF duration of the specific
331
ultrasonic system for the desired application must be determined to get maximum product
332
yield, as unnecessary increase in the ON time would result in the excessive heating and
333
unnecessary electrical energy consumption.
334
3.6. Reusability of enzyme
335
There is a necessity to reuse the biocatalyst after every cycle as enzyme plays a very vital part
336
in the commercial profitability of enzyme catalyzed reactions. Therefore, reusability study
337
was critically vital for the economic feasibility of reaction. Reusability study was carried out
338
using optimized parameters. After each cycle, the immobilized lipase enzyme was filtered off
339
followed by washing with acetone and then dried in oven for 30 min at 40 °C and then kept in
340
the desiccator at room temperature (30 ± 2 °C). The recycled enzyme was used for the next
341
cycle. The results obtained after seven cycles are depicted in Fig. 7(a) which demonstrates
342
that the biodiesel yield generally decreases with an increase in the number of cycles. Also it
343
has been observed that the catalytic activity of immobilized lipase also decreased after each
344
cycle as shown in Fig. 7(b). Only 25% of original enzyme activity was retained after 7 cycles.
345
The obtained results are in well agreement with the Zhang et al. [38] where only 20%
346
conversion was achieved after 6 cycles of reuse for the Lipozyme TLIM enzyme without
347
ultrasonic irradiation. Paludo et al. [39] have also reported that enzyme activity of Lipozyme
348
TLIM decreases completely in only 4 cycles in the presence of mechanical agitation. The
349
decrease in activity and decrease in biodiesel yield may be attributed to the denaturation and
350
breakage of bonding between enzyme and supporting media upon use [40]. Thus it can be
351
established that Lipozyme TLIM activity decreases after repeated use in the biodioesel
352
synthesis reaction. Based on the comparison of the data available in the literature for the
15
353
conventional approach, it is expected that use of ultrasound under the set of optimum
354
conditions does not contribute significantly to enhance the deactivation, especially in the first
355
few cycles of treatment.
356
3.7. Structural characterisation of immobilised lipase
357
The influence of ultrasonic irradiation on the secondary structure of enzyme was analysed by
358
CD spectroscopy. The structural change of the lipase was compared to the ultrasound
359
irradiated enzyme (after use in the multiple cycles) through CD spectra analysis as shown in
360
Fig. 8. Intensity of CD peaks indicates the extent of ellipticity in the protein [41], which
361
shows the changes in protein structure. Ultrasound-treated Lipozyme TLIM showed
362
significant changes in the CD spectra, which reflected the structural changes in protein
363
secondary structure. Compared to non-sonicated enzyme, the amount of α-helix in ultrasound
364
irradiated-immobilized enzyme decreased, whereas β-sheet and unordered polypeptide
365
amount increased as shown in Table 2. Ultrasound irradiated immobilised lipase retained
366
about 79% of its native α- helix content, as obtained from the mean residue ellipticity at 222
367
nm. Application of ultrasound resulted in pressure-induced spectral changes which were
368
indicative of change in the proportion of α-helical and β-sheet structure in the protein. The
369
secondary structure of protein not only depends on the amino acid sequence but also on the
370
interactions between different parts of the molecule [42]. Stathpulos et al. [43] have reported
371
similar findings for the changes in the enzyme structure.
372
3.8. Surface morphology of immobilized lipase
373
The SEM images of the untreated and ultrasound irradiated immobilized lipase samples
374
shown in Fig. 9 revealed that morphological changes were brought about by ultrasonication.
375
The surface of unused enzyme was more or less smooth, while after irradiation, the structure
376
was open though small holes were observed on the surface, which can be attributed to the 16
377
indentations introduced by the cavitational effects. Comparison of SEM images of the
378
immobilized enzyme before and after 7 cycles of use in ultrasound-assisted reaction shows
379
that there is a substantial change in morphology. Thus, ultrasonic treatment caused alterations
380
in Lipozyme TLIM both in terms of the morphology as well as structure at the molecular
381
level. It is important to note that some degree of morphological changes are expected to
382
increase the activity due to the enhanced access of the substrate molecules to the active
383
catalytic sites. Still continuous use, especially under conditions of intense cavitation, would
384
be detrimental contributing to the decrease in the activity of the enzyme. Zhang et al. [42]
385
also reported similar observation for ultrasound irradiated Candida Antarctica lipase B
386
(CalB), where surface was quite different from the initial morphology of lipase without
387
sonication.
388
4. Conclusions
389
Novel
390
interesterification of WCO with methyl acetate has been investigated. Methyl acetate proved
391
to be efficient acyl acceptor giving significant yields of biodiesel. Thus the approach
392
presented in the work avoids the use of methanol and hence possible deactivation of enzyme
393
due to the use of methanol could be avoided. Ultrasound-assisted approach resulted in
394
significant intensification in the process with reduction in reaction from 24 h to 3 h as
395
compared to conventional stirring method. Low frequency mild ultrasonic irradiation was
396
established to be quite important to enhance the rate of enzymatic reaction. The optimum
397
ultrasound parameters observed were 20 kHz frequency, 80 W power with 60% duty cycle.
398
Use of ultrasound also allowed working with lower enzyme loading as compared with
399
conventional approach for giving similar biodiesel yields which also should reduce the cost
400
of production. Overall, it has been established that the interesterification of WCO with
approach
for
ultrasound-assisted
enzyme
(Lipozyme
TLIM)
catalyzed
17
401
methyl acetate using Lipozyme TLIM under ultrasonic irradiation gives an interesting
402
alternative for synthesis of biodiesel with significant process intensification benefits.
403
404
Acknowledgment
405
Authors acknowledge the funding of Department of Science and Technology and Portuguese
406
Science Foundation under the Indo-Portuguese collaborative program (Project Reference:
407
DST/INT/PORTUGAL/P-11/2013)
408
409
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410
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catalyzed hydrolysis of soy oil in solvent-free system, Ultrason. Sonochem. 15 (2008)
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monolaurate from 1,2-propanediol and lauric acid under microwave irradiation: kinetics
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of forward and reverse reactions, Enzyme Microb. Technol. 38 (2006) 814–820.
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and Stability of Immobilized Thermomyces lanuginosus in Producing Fatty Acid Methyl
491
Ester (FAME), IJSRP 3 ( 2013) 1–4.
21
492 493
[32] P. Eisenberg, Cavitation, Handbook of Fluid Mechanics, Tata McGraw Hill Publications, 1984 (Chapter-12).
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understanding cavitational activity distribution and effect of operating parameters, Chem.
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497 498 499 500 501 502
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assisted extraction of antioxidants from pomegranate peel, Ultrason. Sonochem. 18
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(2011) 1249–1257.
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by immobilized Thermomyces lanuginosus lipase (Lipozyme TLIM), Bioprocess
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Biosyst. Eng. 34 (2011) 1163–1168.
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511
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synthesis of glycerol carbonate from glycerol and dimethyl carbonate, Ultrason.
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518
the analysis, Anal. Biochem. 287 (2000) 243–251.
519
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520
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521
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522
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523
Meiering, Sonication of proteins causes formation of aggregates that resemble amyloid,
524
Protein Sci. 13 (2004) 3017‒3027.
525
526
527
23
528
List of Figures
529
Scheme 1. Production of fatty acid methyl esters by enzymatic interesterification of
530
triglyceride with methyl acetate
531 532
Fig. 1. Experimental set up of ultrasound-assisted interesterification reaction
533
Fig. 2(a). Effect of molar ratio on the biodiesel yield for the conventional approach of
534
enzymatic biodiesel production
535
Fig. 2(b). Effect of molar ratio on the biodiesel yield for the ultrasound-assisted approach of
536
enzymatic biodiesel production
537
Fig. 3(a). Effect of enzyme loading on the biodiesel yield for the conventional approach of
538
biodiesel production
539
Fig. 3(b). Effect of enzyme loading on the biodiesel yield for the ultrasound-assisted
540
approach of biodiesel production
541
Fig. 4. Effect of temperature on enzymatic biodiesel yield
542
Fig. 5. Effect of ultrasonic power on the biodiesel yield for ultrasound-assisted approach of
543
enzymatic biodiesel production
544
Fig. 6. Effect of duty cycle on the biodiesel yield for ultrasound-assisted approach of
545
enzymatic biodiesel production
546
Fig. 7(a). Effect of reusability of enzyme on the biodiesel yield for ultrasound-assisted
547
approach of enzymatic biodiesel production
548
Fig. 7(b). Effect of reusability on the relative enzyme activity for ultrasound-assisted
549
approach of enzymatic biodiesel production
550
Fig. 8. CD spectra of unused and reused immobilized lipase
551
Fig. 9. SEM images of: (A) Unused immobilized lipase and (B): Reused immobilized lipase
552
24
553
List of Tables
554
Table 1. Composition and properties of waste cooking oil
555
Table 2. Contents of secondary structure of non-sonicated and sonicated immobilized lipase
556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572
25
573 574 575
Scheme 1: Production of fatty acid methyl esters by enzymatic interesterification of
576
triglyceride with methyl acetate
577
Generator
Horn
Reactor
Water out Reaction mixture Drain
Water in
578 579
Fig. 1. Experimental set up of ultrasound-assisted interesterification reaction
580
26
581 582
Fig. 2(a). Effect of molar ratio on the biodiesel yield for the conventional approach of
583
enzymatic biodiesel production
584 585
Fig. 2(b). Effect of molar ratio on the biodiesel yield for the ultrasound-assisted approach of
586
enzymatic biodiesel production
27
587 588
Fig. 3(a). Effect of enzyme loading on the biodiesel yield for the conventional approach of
589
biodiesel production
590 591
Fig. 3(b). Effect of enzyme loading on the biodiesel yield for the ultrasound-assisted
592
approach of biodiesel production
28
593 594
Fig. 4. Effect of temperature on the biodiesel yield for the conventional approach of
595
enzymatic biodiesel production
596 597
Fig. 5. Effect of ultrasonic power on the biodiesel yield for ultrasound-assisted approach of
598
enzymatic biodiesel production
29
599 600
Fig. 6. Effect of duty cycle on the biodiesel yield for ultrasound-assisted approach of
601
enzymatic biodiesel production
602 603
Fig. 7(a). Effect of reusability of enzyme on the biodiesel yield for ultrasound-assisted
604
approach of enzymatic biodiesel production
605 30
606 607
Fig. 7(b). Effect of reusability on the relative enzyme activity for ultrasound-assisted
608
approach of enzymatic biodiesel production
609
610 611
Fig. 8. CD spectra of unused and reused immobilized lipase
31
(A) Unused immobilized lipase
(B) Reused immobilized lipase
612 613
Fig. 9. SEM images of: (A) Unused immobilized lipase and (B): Reused immobilized lipase
614 615 616 617
32
618
Table 1. Composition and properties of waste cooking oil Property
Value
Linoleic acid (%)
73.4
Oleic acid (%)
18.3
Palmitic acid (%)
6.7
Stearic acid (%)
1.6
Saponification value (mg KOH/g of oil)
198
Density (kg/m3)
930
Acid value (mg KOH/g oil)
4.3
Viscosity (mm2/s)
54.3
619 620
Table 2. Contents of secondary structure of non-sonicated and sonicated immobilized lipase α-Helix
β-Sheet
β-Turn
Random
Relative enzyme
(%)
(%)
(%)
coil (%)
activity (%)
Non-sonicated
52.51
15.43
11.92
18.42
100
Sonicated
41.61
13.62
19.96
22.98
25.1
Treatment
621 622
33
623
Research Highlights
624 625
•
Intensified enzymatic biodiesel production using interesterification approach
626
•
Detailed investigation into the effect of different operating parameters.
627
•
Ultrasonic method gave significant benefits than conventional stirring method.
628
•
Significant reduction in time and requirement of lower loading
629
630 631 632 633 634 635
34