- Email: [email protected]
Process optimization of biodiesel production from Hevea brasiliensis oil using lipase immobilized on spherical silica aerogel
Accepted Manuscript Process optimization of biodiesel production from Hevea brasiliensis oil using lipase immobilized on spherical silica aerogel A. Arumugam, D. Thulasidharan, Gautham B. Jegadeesan PII:
S0960-1481(17)30983-7
DOI:
10.1016/j.renene.2017.10.021
Reference:
RENE 9309
To appear in:
Renewable Energy
Received Date: 20 April 2017 Revised Date:
22 September 2017
Accepted Date: 7 October 2017
Please cite this article as: Arumugam A, Thulasidharan D, Jegadeesan GB, Process optimization of biodiesel production from Hevea brasiliensis oil using lipase immobilized on spherical silica aerogel, Renewable Energy (2017), doi: 10.1016/j.renene.2017.10.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Process optimization of biodiesel Production from Hevea brasiliensis Oil using
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Lipase Immobilized on Spherical Silica Aerogel
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1. Dr. A. Arumugam,
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2. Mr. Thulasidharan.D
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3. Dr. Gautham B. Jegadeesan
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School of Chemical &Biotechnology,
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SASTRAUNIVERSITY,
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Thirumalaisamudram, Thanjavur - PIN: 613 401, INDIA.
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Email: [email protected].
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Process optimization of biodiesel Production from Hevea brasiliensis Oil using
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Lipase Immobilized on Spherical Silica Aerogel
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Arumugam. A*,Thulasidharan. D, Gautham B. Jegadeesan
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School of Chemical & Biotechnology, SASTRA University, Thanjavur, India - 613401.
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Email: [email protected]
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ABSTRACT
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In this study, biodiesel was synthesized in an enzymatic transesterification process from Hevea
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brasiliensis, crude non-edible oil, using lipase immobilized on spherical silica aerogels.
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Enzymatic transesterification is preferred to chemical methods as it is milder and is more
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environmentally friendly. Lipase based transesterification of Hevea brasiliensis under optimal
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conditions provided high FAME (fatty acid methyl esters) yields up to 93%. Response Surface
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Methodology (RSM) was used to optimize the process for maximum FAME yield.
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maximum yield was obtained at a temperature of 35°C, water content of 15% (v/v %) and
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methanol/oil molar ratio of 8:1. Percent yields of FAME from the transesterification process
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followed second order model. Even after 10 cycles of reuse, lipase immobilized on spherical
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silica aerogel showed only 10.7 % reduction in percentage yield of FAME. The results from this
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study demonstrate the viability of economical biodiesel production using waste products as both
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source and catalyst.
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Keywords: Biodiesel, Transesterification, Hevea brasiliensis (Rubber seed) oil, Lipase,
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Mesoporous Silica aerogel, Response surface methodology (RSM).
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The
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1.
INTRODUCTION
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Biodiesel has been generally accepted and defined as a substitute for or an additive to diesel fuel,
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which is derived from natural sources [1]. Sources for biodiesel production are plenty mostly
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from oils and fats of plants and animals derived from a renewable lipid feedstock [2]. The main
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advantages of using this alternative fuel is its renewability, lower gaseous emissions, and its
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biodegradability. Since all the organic carbon present has a photosynthetic origin, there is no net
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increase in CO2 levels in the atmosphere [3]. Given the food scarcity in several regions around
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the world, there is an increased focus on the use of non-edible oils for biodiesel production.
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Some of the non-edible feed stock available and reported are Barbados nut (Jatropha curcus) [4],
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Neem (Azadirachta indica) [5], Castor (Ricinus communis)[6], Linseed (Linumus itatissimum)
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[7], Karanja (Pongamia pinnata)[8] and Pinnai (Calophyllum inophyllum)[9].
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The two main challenges to large-scale biodiesel production are: (1) selection of non-edible
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feedstock with high oil content and their abundance; and (2) synthesis method (chemical or
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enzymatic). Hevea brasiliensis, commonly known as rubber seed, is one such low cost non-
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edible feedstock, which is found in abundance in the Amazon [10]. The Hevea brasiliensis seed
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contains high oil content (up to 89.4%) and approximately 80.5% of the oil is in the form of
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unsaturated fatty acids (essentially linoleic and oleic acids). The oil extracted from the seed is
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blackish brown in colour with an unpleasant aromatic odour [11, 12]. Given the high oil content,
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there has been increased recent focus on using this feedstock for biodiesel production, as
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summarized in Table 1. As can be seen in Table 1, the focus of most studies was to evaluate the
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biodiesel yield from rubber seed oil. In most studies, chemical catalysts (homogenous and
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heterogeneous) are used [13, 14,15,16]. Use of acid catalysts such as H2SO4 and base catalysts
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such as KOH [18; 13] has shown that Hevea brasiliensis oil transesterification yields 31-99%
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methyl esters, depending upon the conditions. Dhawane and his co-workers reported almost
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90% yields at an optimum molar ratio of 15:1, 55°C, reaction time of 60 minutes and 3.5% (w/w
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of oil) catalyst loading. The study also revealed that after three cycles of reuse, there was only
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1.4 to 1.8% reduction in percentage conversion [18].
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sulfonic acid-functionalised MCM-41 as catalyst showed 96% FAME (fatty acid methyl esters)
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yield at 5% catalyst loading, 120 min reaction time and 153 °C reaction temperature [15].
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Widayata and his co-workers produced 91% biodiesel yield from rubber seed using H2SO4
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catalyst (0.1 to 1% of catalyst loading, 0.5 (v/v%), 1:1.5- 1:3 quantity of methanol/oil (molar),
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and a reaction time of 120 h [19].
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The other challenge, as noted earlier, is the method of transesterification. Chemical
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transesterification of non-edible oils to biodiesel is a preferred option because of its flexibility in
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use, large production rates and capacities, and high yields [20, 21]. However, the high costs,
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post-synthesis environmental issues and high energy requirements warrant the need to search
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alternate technologies. As noted earlier, most studies on transesterification of Hevea brasiliensis
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oil used chemical catalytic methods. However, the chemical catalysed transesterification process
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requires reaction temperature of at least 60°C to 80°C, high methanol to oil molar ratio of 12:1 to
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36:1 and multi stage pre-treatment processes to reduce the free fatty acid content. These
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drawbacks can be eliminated by enzymatic transesterification, which offer a promising
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alternative to the chemical methods. Enzymatic transesterification offers the advantages of low
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temperature and pressure conditions (ambient), high catalyst recyclability and equally good
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FAME yields. In our previous works [9, 21], we have successfully demonstrated that use of
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immobilized enzyme for transesterification process which is not only viable, but also a better
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alternate to chemical transesterification.
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Another study using methyl propyl
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To the best of our knowledge, there has been no work till date on the production of biodiesel
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using Hevea brasiliensis oil via enzymatic transesterification process. This work is the first of its
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kind to be reported on biodiesel production from crude Hevea brasiliensis oil (CHBO) catalyzed
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by immobilized lipase spherical silica aerogel. Immobilized enzymes are used to: (1) reduce the
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cost of the enzyme; (2) improve catalyst stability and recyclability; and (3) prevent reaction
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inhibition due to high substrate and product concentrations. Further, this process seeks to
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improve activity and selectivity which is challenging for commercialization of lipase-catalyzed
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biodiesel production. The specific objectives of the study are: (1) optimization of the process
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parameters such as molar ratio of substrates, water content and temperature on yield; and (2)
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reusability of the catalyst for efficient biodiesel production. Optimization of the important
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process variables namely molar ratio of methanol to oil (3:1 – 8:1), volume of water (5%v/v -
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25% v/v) used and reaction temperature (30°C-40°C) maintained were done using Response
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Surface Methodology (RSM).
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2.
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The lipase (E.C.3.1.1.3) used in the present study is the commercial Lipase (purity 99%, activity
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16000 LU/g was purchased from Himedia Pvt. Ltd.The crude Hevea brasiliensis (rubber seed)
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oil (Average molecular weight, 861.4 g mol-1, specific gravity, 0.919) was obtained from nearby
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agricultural field in Thanjavur, India. Coal bottom ash is obtained from NLC India Limited
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(Tamilnadu, India). Sodium hydroxide pellets (99%, Merck), ethanol (95%, Merck),
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hydrochloric acid (98%, Merck) are obtained from Merck Chemicals Pvt. Ltd. Methanol,
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dipotassium hydrogen phosphate, copper sulphate pentahydrate, lipase, sodium potassium
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tartarate, potassium dihydrogen phosphate, sodium carbonate, Folin‘sciocalteau reagent and gum
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arabic have been purchased from Himedia Laboratories Pvt. Ltd.
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MATERIALS AND METHODS
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2.1. Preparation of silica aerogel and Lipase immobilization
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Silica aerogel microspheres are prepared following the procedure reported in literature [22]. 0.1
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g of spherical aerogel particles was dispersed in 10 ml of potassium phosphate buffered to pH of
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7. Precisely weighed lipase (10 mg) was added to the above mixture and stirred for 12 h,
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maintaining temperature to 30 ºC. The immobilized lipase was separated by filtration and the
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percentage immobilization and specific enzyme activity were determined. The enzyme
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concentration was measured by Lowry method [23]. Specific enzyme activity for immobilised
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enzyme was estimated by Olive oil emulsion method [24]. The percentage immobilization,
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specific enzyme activity of the immobilized lipase on silica aerogel was 77% and 14800 U/ g.
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2.2. Transesterification reaction
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The transesterification reaction was carried out in a 100 mL screw capped vessel using 20 gm of
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CHBO, 15 % (v/v %) water content, 430 mg immobilized lipase and at a constant temperature of
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30˚C. Typically, higher water content ensures higher enzymatic activity [25]. However, the water
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content was maintained at this level to: (1) prevent hydrolysis of the ester linkages at even higher
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water content, thereby allowing for the forward transesterification reaction [26]; and (2) ensure
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insignificant mass transfer interferences between the aqueous and oil phase (since enzyme lipase
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have a unique feature to act at the interface between the aqueous and organic phases).
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Methanol was added to the reaction vessel in a three step process at 1:8 molar ratio of
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oil/methanol. For example, 2.72 ml of methanol was added to 22.5 mL of oil, which
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corresponded to a molar ratio of 1:3. To increase the methanol: oil molar ratio to 1:9, 8.15 mL of
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methanol was added to 22.5 mL of oil, while keeping the amount catalyst at 430 mg. The
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reaction was carried out in a shaking incubator at 180 oscillations per minute. After a 10 h
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reaction, 100 ml of sample was taken from the reaction mixture and centrifuged. The upper layer
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was analysed in GC-MS [27]. All the experiments were done in triplicates.
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2.3 Response Surface Methodology (RSM)
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Various experiments have been conducted to identify the important process variables, their effect
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on the response variables and the interaction between the variables. Response Surface Method is
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a statistical experimental design that helps identify optimum process conditions with less number
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of experimental runs as compared to conventional experimental design. Thus, cost of expensive
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experimental runs is minimized by RSM [28 - 29]. The main advantage in using RSM is the
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speed and reliability of the outcome. Contours are response curves drawn in 2-dimensional
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plane, keeping other variables fixed [30]. The shape of contour plots of the response helps us to
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visualize interaction between the variables. A quadratic model was employed to express the
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response variable in terms of the independent variables. Later the model was solved analytically
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to obtain the optimum conditions [28]. All the statistical analyses were conducted using Minitab
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statistics software (Version 16.2.2, Minitab Inc, Pennsylvania, USA). Analysis of variance
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(ANOVA) was performed on the data to test the effects of the parameters and their interactions.
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Tukey's multiple tests were performed to determine the differences among the levels of each
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parameter. The α-level chosen was 0.05. The RSM utilized Taylor first order and second order
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series with experimental data for optimization. The surface of Taylor expansion curve was
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determined using RSM and this describes the response. It is of the form (Eqs.1):
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Re sponse = β 0 + ∑ β i x i + ∑ β ii x 2 + ∑ β ij x i x j
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Where,
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(1)
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βi, βij = Regression coefficients
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x= Process variable
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2.3 Catalyst and product characterization
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The synthesized catalyst matrix was characterized using Scanning Electron Microscope imaging
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for surface morphology (6701 F, JEOL, Japan). Brunauer-Emmett-Teller (BET) method was
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used to determine specific surface area of support matrix. Barrett-Joyner-Halenda model (BJH)
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was used to determine pore size distribution. N2 gas adsorption technique was used to determine
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volume of pores, average pore diameter and surface area of support matrixes. Methyl esters
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formed during the experiment were examined by Gas Chromatography–Mass Spectroscopy
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(CLARUS 500, PerkinElmer, USA). Fourier transform infrared spectroscopy (spectrum 100,
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Perkin Elmer, USA) was used to characterize the functional group attached on mesoporous
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silica. Quantification of methyl ester content in the reaction mixture was carried out using
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(0.1mm x 10cm) gas chromatography capillary column. The column temperature was kept at 180
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°C for 0.5 min, raised to 300 °C at 10 °C min-1 and maintained at this temperature for 10 min.
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The temperatures of the injector and detector were set at 245 °C and 305 °C, respectively. The
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fuel properties such as density, flash point, fire point, pour point, cloud point, kinematic
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viscosity, Cetane number, Calorific value of biodiesel obtained from CHBO were measured
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using ASTM methods.
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3. RESULT AND DISCUSSION
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3.1 Characterization
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SEM image of silica aerogel (Figure 1) shows that the synthesized aerogel particles are in the
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form of solid clusters with size ranging from 22 to 25 nm [31]. The BET surface area, average
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pore volume and average pore diameter were determined to be 443 m² g-1, 0.19 mL g-1 and 2.88
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nm respectively (Supplementary Figure 1a & 1b). Figure 2 shows the FT-IR spectrum for the
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bare and lipase immobilized silica aerogel. The presence of a broad band at about 3471 cm−1,
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corresponding to the vibration of Si-OH terminal groups. An absorption peak at 1108 cm−1
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corresponds to asymmetric stretching vibrations of siloxane bond Si–O–Si. The FT-IR spectrum
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of silica aerogel after immobilization confirmed the presence of a number of functional groups in
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the enzyme structure. Signal at 1661 and 1542 cm-1 generated by the N-H bending is the
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characteristic for the pure enzymes corresponds to amide 1 and amide II bands [32]. Contribution
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of the C–N stretching vibrations is more likely due to amide III (1426 cm−1) bands resulting from
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N–H bending. Water is critical for enzymes structures, conformation and interaction with solid
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hydrophilic surfaces. The 2961 cm-1 bands may be assigned to -OH bonded to the protein [33].
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There was no change in spectrum frequency of surface functional group and disappearance of
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any peak of silica aerogel, suggesting physical adsorption of the enzyme on the solid matrix.
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3.2 Effect of oil-alcohol ratio on reaction (MR)
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The rate of the reaction depends on the alcohol to oil mole ratio in the reaction mixture. If the
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alcohol concentration is increased, an increased reaction rate can be observed. But too high
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methanol concentration leads to deactivation of enzyme. Addition of alcohol in regular intervals
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to maintain low concentration can improve the enzyme catalysis and will prevent the
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denaturation. Figure 3 shows the variation of methanol to oil molar ratio on percentage yield of
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biodiesel. The yield of biodiesel increases from 3:1 to 6:1 and reaches maximum yield of 93% at
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8:1 and then decreases. Increasing trend in the biodiesel yield is due to higher concentration of
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methanol and decreasing yield might be due to the denaturation of the immobilized lipase [34].
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199 3.3 Effect of Temperature (T)
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Biodiesel production by enzymatic method requires milder conditions which is less energy
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intensive when compared to the chemical method. It is well known that rates of most reactions
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(enzymatic or chemical) tends to increase with temperature, and usually expressed by their
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Arrhenius constant. In this study also, it was observed that the rate increased with temperature.
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From the Figure 4, it was observed that the yield of biodiesel is highest (92%) at the 30°C [35].
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However, most studies have limited their range of temperature investigated to below 40 °C for
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two reasons: (1) Increasing temperature above 40 °C may denature the enzyme, decreasing the
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rate of reaction; (2) good yields can be obtained at lower temperatures [36]. It was observed that
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increase in temperature beyond 30°C caused the decline in the yield of biodiesel (Figure. 4).
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Increasing the temperature from 20°C to 30°C showed a sudden rise in biodiesel yield owing to
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higher reaction rate. As the temperature increases beyond 35°C the yield of biodiesel reduces due
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to the loss of the enzyme activity [37]. It has been noted that lower temperature slows down the
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rate, thereby prolonging the reaction time required to produce a similar yield. Increasing the
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reaction temperature may lower yield due to the reversible nature of the reaction, and loss of
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active sites due to enzyme denaturation [38].
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3.1 Optimization parameters for biodiesel production
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In this study, 3-level-3-factor design was implemented, totally 20 experiments were done in
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duplicates [39]. The prophase research results showed that the three independent variable
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parameters such as reaction temperature (T), molar ratios of methanol to oil (MR) and water
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content (W) have important effects on FAME yield [33,34]. Water content (v/v) was 5-25%;
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molar ratios of methanol to oil (mol/mol) was 3:1–8:1 and the reaction temperature was between
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30 and 40 (°C). The feedback was FAME yield in percentage (FAME). The non-dependent
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factors, levels, and experimental model are tabulated in Table 2 and Table 3 [40, 41].
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The model predicted was correlated to coefficients of interactions, linear and quadratic effects.
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The correlation coefficients for each model and variable significance was measured by the
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probability values are shown in Table 4.All the factors and their square interactions (P < 0.05)
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except interaction term of methanol to oil molar ratio and water content were significant at the
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The best fit for the experimental data (Table 2), expressed by polynomial model for the
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percentage yield of FAME is as follows:
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− 0 .33 T 2 + 0 .202 M R2 − 0 .167 × W
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− 0 .0159 T × W + 0 .000313 M R × W
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The R2 value determines the amount of variability in the observed response values that could be
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described by the experimental factors and their interactions [41]. Adj-R2 value of 99.82% was
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observed. As both R2 and Adj-R2 values are high and close to 1.0, a high link between the
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observed and predicted values can be observed. This proves that the regression model provides
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exceptional data on the relationship between independent variables and the response validation
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of the model [42]. The optimum level of percentage yield of FAME was 94.33% at methanol to
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oil molar ratio of 8:1, reaction temperature of 35ºC and 15% water content (v/v).
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3.4 Reusability studies of immobilized lipase
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The reusability of lipase immobilized on spherical silica aerogel was investigated by recovering
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the immobilized particles after each reaction cycle (Figure 5). Approximately 90% of the activity
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(in terms of methyl ester formation) was retained after 10 cycles of reaction. From the Figure. 5
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it is evident that the gradual decrease in FAME yield was attributed to both loss of activity of
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immobilized lipase and loss of enzyme due to leaching. Lipase immobilized on silica aerogel can
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be reused repeatedly without significant loss in activity in the production of biodiesel from
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CHBO [43].
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3.5 Fuel properties
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Fuel properties of crude Hevea brasiliensis oil were determined by ASTM methods. Values of
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flash point, fire point, pour point and cloud point were 149 ± 0.52°C, 191.6 ± 0.89°C, -4 ±
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0.026°C, and -2 ± 0.10°C, respectively. Calorific value and Cetane number (38°C) of the
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biodiesel produced were 37.9 (MJ/Kg) and 52 respectively. Kinematic viscosity and specific
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gravity (38°C) of the biodiesel produced from rubber seed oil were 4.97 ± 0.35 (mm2/sec) and
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0.831 ± 0.02 (g/cm3). Fuel properties of rubber seed Biodiesel were found to be compatible with
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ASTM Biodiesel Standard (D 6751a) and European Biodiesel Standards (EN 14214). This
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demonstrates the feasibility of crude Hevea brasiliensis oil biodiesel as fuel.
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In this work, we have demonstrated the production of biodiesel using Hevea brasiliensis, in an
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immobilized lipase based transesterification process. The optimal condition for methanolysis was
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8:1 molar ratio of methanol to oil and reaction temperature of 30°C. Under these conditions, 93
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% yield of methyl ester were obtained. RSM based studies suggested a second order model, with
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yield dependent on temperature, pH and methanol to oil molar ratio. The fuel produced from the
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lipase based transestrification process was found compatible with ASTM Biodiesel Standard (D
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6751a) and European Biodiesel Standards (EN 14214). Effective use of a waste material as a
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support material for the enzymatic reaction helps reduce the production cost. The results suggests
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strongly that enzymatic transesterification of triglycerides offer an more environmentally
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approach to biodiesel production, with potential for direct use of the product in diesel engines.
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High FFA Jatropha Curcas Oil Using Response Surface Methodology. Sci. 1 (2012) 36-43.
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canola oil catalyzed by various lipases immobilized on epoxy-functionalized silica as low cost
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Vazquez ,Irene Suarez-Marcos. Biodiesel Production Over Arenesulfonic Acid-Modified
390
Mesostructured Catalysts: Optimization of Reaction Parameters Using Response Surface
391
Methodology. Top Catal. 53 (2010) 795–804.
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water-resistant catalyst. Bioresource. Technol. 102 (2011) 2665-2671.
406
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[52] Vc. Vipin., J. Sebastian, C. Muraleedharan, A. Santhiagu, Enzymatic transesterification of
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6. List of figures and tables
417
Figure 1. SEM image of Silica aerogel.
418
Figure 2 FTIR spectra of bare and immobilized silica aerogel material.
419
19
ACCEPTED MANUSCRIPT
Figure 3: Effect of Methanol to oil molar ratio on immobilized lipase catalyzed methanolysis of
421
Hevea brasiliensis oil for Temperature 30ºC, 10% v/v water content and reaction time of 10 h.
422
Figure 4: Effect of temperature on immobilized lipase catalyzed methanolysis of Hevea
423
brasiliensis oil for 8: 1 molar ratio of methanol to oil, 10%v/v water content and reaction time of
424
10 h.
425
Figure 5: Immobilized lipase catalyzed methanolysis of Hevea brasiliensis oil for several cycles
426
at optimum conditions (8: 1 molar ratio of methanol to oil, 10%v/v water content, Temperature
427
30ºC and reaction time of 10 h).
428
Table 1: The comparison of biodiesel production from Hevea brasiliensis using various catalyst
429
in the literature with the present work.
430
Table 2: Experimental results based on central composite design.
431
Table 3: Estimated Regression Coefficients for yield of FAME
432 433
Table 4: Analysis of Variance for yield
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ACCEPTED MANUSCRIPT Table 1: The comparison of biodiesel production from Hevea brasiliensis using various catalyst in the literature with the present work.
Operating parameter’s
Lipase from Aspergillus niger
8:1 molar ratio of methanol to oil, 15 % water content and 35°C, 430 mg immobilized lipase/g oil, reaction time 7 h. Methanol to oil molar ratio of 3:1– 15:1, a catalyst concentration of 0.25– .25%wt., a reaction temperature of 50–70 °C, a reaction time of 30–150 min and speed agitation of 800–1200 rpm
93
SC
Acid esterification: KOH
% yield
Work
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Catalyst
Present work
A.S. Silitonga et al, 2016 [13]
31
Oryzae Lipase
Methanol to oil molar ratio of 1:4 and catalyst concentration of 15(w/w) % of oil after 48 h.
V.V. Vipin et al, 2016 [52]
Activated carbon impregnated with pure KOH
Reaction temperature 55 °C, Reaction time 60 min, catalyst loading 3.5 wt% and methanol to oil ratio 15:1.
89.81
Sumit H. Dhawane et al, 2015 [14]
84
S. Karnjanakom et al, 2015 [15]
Temperature 60 °C, reaction time 1 h, and 5 g of carbon based catalyst at varying quantities of catalyst loading (0.5, 2, 3.5, 5 wt%) and methanol to oil ratio (5:1–20:1)
89.3
Sumit H. Dhawane et al, 2015 [16]
6:1 alcohol to oil ratio, 1% catalyst concentration, 55 °C reaction temperature and 67.5 min reaction time.
96.8
Junaid Ahmad et al, 2014 [20]
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SO3H-MCM-41
Methanol to oil ratio 16:1,Catalyst loading of 14.5 wt.% and a reaction time and temperature of 48 h and 129.6°C,
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Rhizopus
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Base transesterification: H2SO4
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99.32
Carbon based KOH impregnated heterogeneous catalyst from flamboyant pods (Delonix regia) Acid esterification: H2SO4
Base transesterification: KOH
ACCEPTED MANUSCRIPT Catalyst concentration in range 0.11%(v/v), raw material to methanol molar ratio (1:2) , Temperature 60 °C , ratio of raw material to methanol in range 1:1.5-1:3 and reaction time 60 min. Catalyst loading of 5 wt. %; methanol to oil molar ratio of 4:1; reaction temperature of 65°C and reaction time of 4 h
Alkaline esterification: NaOH Base transesterification: NaOH Alkaline esterification: H2SO4
NaOH
96.9
SC
Methanol/oil molar ratio of 9:1 and 0.5% by weight of sodium hydroxide
1% catalyst loading, 6:1 alcohol to oil ratio, 97.8 ± 0.2 C °C reaction temperature and 60 min reaction time.
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NaOH
Methanol/oil ratio 0.28 % v/v, sodium hydroxide of 0.75% w/v, Temperature 51.23 °C, Reaction time 82.52 min
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Base transesterification:
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Poly (sodium acrylate) supported NaOH
Methanol/oil molar ratio 6:1, stirring speed 1000 rpm, reaction temperature 60 °C, 3 wt% NaOH/NaPAA sample with NaOH loading amount of 7.5 mmol/g was used as catalyst
TE D
Limestone based catalyst
91.05
Widayat et al, 2013 [19]
RI PT
H2SO4
Jolius Gimbun et al, 2013[47]
96
Ru Yang et al, 2013 [48]
97.1
D.F. Melvin Jose et al, 2013 [49]
-
A.S. Ramadhas et al, 2005 [50]
84
O.E. Ikwuagwu et al, 2000 [51]
ACCEPTED MANUSCRIPT Table 2: Experimental results based on central composite design. Pred
Blocks
T
MR
W
% yield
% yield
1
1
1
30
4
5
66.30
66.40
2
2
1
1
40
4
5
59.30
59.18
3
3
1
1
30
8
5
69.10
68.99
4
4
1
1
40
8
5
71.20
71.07
5
5
1
1
30
4
25
64.67
64.82
6
6
1
1
40
7
7
1
1
30
8
8
1
1
9
9
-1
1
10
10
-1
1
11
11
-1
1
12
12
-1
13
13
-1
14
14
-1
15
15
16
RI PT
1
SC
StdOrder RunOrder PtType
Exp
25
54.28
54.41
8
25
67.30
67.44
40
8
25
66.40
66.33
30
6
15
83.21
82.82
40
6
15
78.61
78.65
35
4
15
86.55
86.19
TE D
M AN U
4
35
8
15
93.44
93.44
1
35
6
5
73.76
73.88
1
35
6
25
71.20
70.72
0
1
35
6
15
89.20
89.01
16
0
1
35
6
15
88.45
89.01
17
0
1
35
6
15
89.40
89.01
18
0
1
35
6
15
88.12
89.01
19
19
0
1
35
6
15
89.77
89.01
20
20
0
1
35
6
15
88.66
89.01
18
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17
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1
ACCEPTED MANUSCRIPT Table 3: Estimated Regression Coefficients for yield of FAME
Term
Coef
SE
T
P
Coef 89.0349
0.1763
505.067
0.000
T
-2.0792
0.1622
-12.822
0.000
MR
3.6340
0.1622
22.410
0.000
W
-1.5810
0.1622
-9.750
0.000
T*T
-8.2784 0.8076
T*W
SC
0.000
0.3092
2.612
0.026
0.3092
-54.030
0.000
0.1813
12.817
0.000
-0.7988
0.1813
-4.406
0.001
0.0062
0.1813
0.034
0.973
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MR *W
-26.772
2.3238
EP
T* MR
16.7074
0.3092
TE D
W*W
M AN U
MR * MR
RI PT
Constant
S = 0.512786 R-Sq = 99.90%
PRESS = 7.25860 R-Sq(pred) = 99.73%
R-Sq(adj) = 99.82%
ACCEPTED MANUSCRIPT Table 4: Analysis of Variance for yield
Source
Seq SS
Adj SS
Adj MS
F
Regression
9
2698.97
2698.97
299.885
1140.47
0.000
Linear
3
200.29
200.29
66.762
253.90
0.000
T
1
43.23
43.23
43.231
164.41
0.000
MR
1
132.06
132.06
132.060
502.22
0.000
W
1
25.00
25.00
24.996
95.06
0.000
Square
3
2450.38
2450.38
816.793
3106.28
0.000
T*T
1
1587.44
188.46
188.461
716.72
0.000
MR * MR
1
95.31
1.79
1.794
6.82
0.026
W*W
1
767.62
767.62
767.624
2919.29
0.000
Interaction
3
48.30
48.30
16.101
61.23
0.000
T* MR
1
43.20
43.20
43.199
164.28
0.000
T*W
1
5.10
5.10
5.104
19.41
0.001
MR *W
1
0.00
0.00
0.000
0.00
0.973
0.34
0.868
Lack-of-Fit
Total
SC
M AN U
TE D 2.63
2.63
0.67
0.67
0.134
5
1.96
1.96
0.392
19
2701.60
P
0.263
5
AC C
Pure Error
10
EP
Residual Error
RI PT
DF
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
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Figure 1. SEM image of Silica aerogel.
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Figure 2 FTIR spectra of bare and lipase immobilized silica aerogel material.
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100
RI PT SC
60
40
M AN U
% yield of biodiesel
80
20
0 4
6
TE D
2
8
10
12
EP
Methanol to oil molar ratio
Figure 3: Effect of Methanol to oil molar ratio on immobilized lipase catalyzed methanolysis of
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Hevea brasiliensis oil for Temperature 30ºC, 10% v/v water content and reaction time of 10 h.
ACCEPTED MANUSCRIPT
100
RI PT SC
60
40
20
0 20
25
30
35
40
TE D
15
M AN U
% Yield of biodiesel
80
Temperature (°C)
EP
Figure 4: Effect of temperature on immobilized lipase catalyzed methanolysis of Hevea
10 h.
AC C
brasiliensis oil for 8: 1 molar ratio of methanol to oil, 10%v/v water content and reaction time of
ACCEPTED MANUSCRIPT
100
RI PT SC
60
40
20
0 1
2
3
4
5
6
7
8
9
10
11
TE D
0
M AN U
% Yield of biodiesel
80
EP
Number of cycles
Figure 5: Immobilized lipase catalyzed methanolysis of Hevea brasiliensis oil for several cycles
AC C
at optimum conditions (8: 1 molar ratio of methanol to oil, 10%v/v water content, Temperature 30ºC and reaction time of 10 h). .
ACCEPTED MANUSCRIPT
Research highlight •
Biodiesel was produced from Hevea brasiliensis by lipase immobilized on a low cost
•
Ordered mesoporous spherical silica aerogel could be secured from cheaper silica precursor originating from coal bottom ash.
The immobilized enzyme, when reused, showed highly effective methanolysis for ten
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•
cycles.
Use of immobilized silica aerogel for biodiesel production was more green and cost
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support.
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effective.
S0960-1481(17)30983-7
DOI:
10.1016/j.renene.2017.10.021
Reference:
RENE 9309
To appear in:
Renewable Energy
Received Date: 20 April 2017 Revised Date:
22 September 2017
Accepted Date: 7 October 2017
Please cite this article as: Arumugam A, Thulasidharan D, Jegadeesan GB, Process optimization of biodiesel production from Hevea brasiliensis oil using lipase immobilized on spherical silica aerogel, Renewable Energy (2017), doi: 10.1016/j.renene.2017.10.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Process optimization of biodiesel Production from Hevea brasiliensis Oil using
2
Lipase Immobilized on Spherical Silica Aerogel
4
1. Dr. A. Arumugam,
5
2. Mr. Thulasidharan.D
6
3. Dr. Gautham B. Jegadeesan
7
School of Chemical &Biotechnology,
8
SASTRAUNIVERSITY,
9
Thirumalaisamudram, Thanjavur - PIN: 613 401, INDIA.
11
Email: [email protected].
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.
15 16 17 18 19
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Authors names and affiliations:
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Process optimization of biodiesel Production from Hevea brasiliensis Oil using
22
Lipase Immobilized on Spherical Silica Aerogel
23
Arumugam. A*,Thulasidharan. D, Gautham B. Jegadeesan
24
School of Chemical & Biotechnology, SASTRA University, Thanjavur, India - 613401.
25
Email: [email protected]
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20 21
ABSTRACT
27
In this study, biodiesel was synthesized in an enzymatic transesterification process from Hevea
28
brasiliensis, crude non-edible oil, using lipase immobilized on spherical silica aerogels.
29
Enzymatic transesterification is preferred to chemical methods as it is milder and is more
30
environmentally friendly. Lipase based transesterification of Hevea brasiliensis under optimal
31
conditions provided high FAME (fatty acid methyl esters) yields up to 93%. Response Surface
32
Methodology (RSM) was used to optimize the process for maximum FAME yield.
33
maximum yield was obtained at a temperature of 35°C, water content of 15% (v/v %) and
34
methanol/oil molar ratio of 8:1. Percent yields of FAME from the transesterification process
35
followed second order model. Even after 10 cycles of reuse, lipase immobilized on spherical
36
silica aerogel showed only 10.7 % reduction in percentage yield of FAME. The results from this
37
study demonstrate the viability of economical biodiesel production using waste products as both
38
source and catalyst.
39
Keywords: Biodiesel, Transesterification, Hevea brasiliensis (Rubber seed) oil, Lipase,
40
Mesoporous Silica aerogel, Response surface methodology (RSM).
41
The
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1.
INTRODUCTION
43
Biodiesel has been generally accepted and defined as a substitute for or an additive to diesel fuel,
44
which is derived from natural sources [1]. Sources for biodiesel production are plenty mostly
45
from oils and fats of plants and animals derived from a renewable lipid feedstock [2]. The main
46
advantages of using this alternative fuel is its renewability, lower gaseous emissions, and its
47
biodegradability. Since all the organic carbon present has a photosynthetic origin, there is no net
48
increase in CO2 levels in the atmosphere [3]. Given the food scarcity in several regions around
49
the world, there is an increased focus on the use of non-edible oils for biodiesel production.
50
Some of the non-edible feed stock available and reported are Barbados nut (Jatropha curcus) [4],
51
Neem (Azadirachta indica) [5], Castor (Ricinus communis)[6], Linseed (Linumus itatissimum)
52
[7], Karanja (Pongamia pinnata)[8] and Pinnai (Calophyllum inophyllum)[9].
53
The two main challenges to large-scale biodiesel production are: (1) selection of non-edible
54
feedstock with high oil content and their abundance; and (2) synthesis method (chemical or
55
enzymatic). Hevea brasiliensis, commonly known as rubber seed, is one such low cost non-
56
edible feedstock, which is found in abundance in the Amazon [10]. The Hevea brasiliensis seed
57
contains high oil content (up to 89.4%) and approximately 80.5% of the oil is in the form of
58
unsaturated fatty acids (essentially linoleic and oleic acids). The oil extracted from the seed is
59
blackish brown in colour with an unpleasant aromatic odour [11, 12]. Given the high oil content,
60
there has been increased recent focus on using this feedstock for biodiesel production, as
61
summarized in Table 1. As can be seen in Table 1, the focus of most studies was to evaluate the
62
biodiesel yield from rubber seed oil. In most studies, chemical catalysts (homogenous and
63
heterogeneous) are used [13, 14,15,16]. Use of acid catalysts such as H2SO4 and base catalysts
64
such as KOH [18; 13] has shown that Hevea brasiliensis oil transesterification yields 31-99%
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methyl esters, depending upon the conditions. Dhawane and his co-workers reported almost
66
90% yields at an optimum molar ratio of 15:1, 55°C, reaction time of 60 minutes and 3.5% (w/w
67
of oil) catalyst loading. The study also revealed that after three cycles of reuse, there was only
68
1.4 to 1.8% reduction in percentage conversion [18].
69
sulfonic acid-functionalised MCM-41 as catalyst showed 96% FAME (fatty acid methyl esters)
70
yield at 5% catalyst loading, 120 min reaction time and 153 °C reaction temperature [15].
71
Widayata and his co-workers produced 91% biodiesel yield from rubber seed using H2SO4
72
catalyst (0.1 to 1% of catalyst loading, 0.5 (v/v%), 1:1.5- 1:3 quantity of methanol/oil (molar),
73
and a reaction time of 120 h [19].
74
The other challenge, as noted earlier, is the method of transesterification. Chemical
75
transesterification of non-edible oils to biodiesel is a preferred option because of its flexibility in
76
use, large production rates and capacities, and high yields [20, 21]. However, the high costs,
77
post-synthesis environmental issues and high energy requirements warrant the need to search
78
alternate technologies. As noted earlier, most studies on transesterification of Hevea brasiliensis
79
oil used chemical catalytic methods. However, the chemical catalysed transesterification process
80
requires reaction temperature of at least 60°C to 80°C, high methanol to oil molar ratio of 12:1 to
81
36:1 and multi stage pre-treatment processes to reduce the free fatty acid content. These
82
drawbacks can be eliminated by enzymatic transesterification, which offer a promising
83
alternative to the chemical methods. Enzymatic transesterification offers the advantages of low
84
temperature and pressure conditions (ambient), high catalyst recyclability and equally good
85
FAME yields. In our previous works [9, 21], we have successfully demonstrated that use of
86
immobilized enzyme for transesterification process which is not only viable, but also a better
87
alternate to chemical transesterification.
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To the best of our knowledge, there has been no work till date on the production of biodiesel
89
using Hevea brasiliensis oil via enzymatic transesterification process. This work is the first of its
90
kind to be reported on biodiesel production from crude Hevea brasiliensis oil (CHBO) catalyzed
91
by immobilized lipase spherical silica aerogel. Immobilized enzymes are used to: (1) reduce the
92
cost of the enzyme; (2) improve catalyst stability and recyclability; and (3) prevent reaction
93
inhibition due to high substrate and product concentrations. Further, this process seeks to
94
improve activity and selectivity which is challenging for commercialization of lipase-catalyzed
95
biodiesel production. The specific objectives of the study are: (1) optimization of the process
96
parameters such as molar ratio of substrates, water content and temperature on yield; and (2)
97
reusability of the catalyst for efficient biodiesel production. Optimization of the important
98
process variables namely molar ratio of methanol to oil (3:1 – 8:1), volume of water (5%v/v -
99
25% v/v) used and reaction temperature (30°C-40°C) maintained were done using Response
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Surface Methodology (RSM).
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2.
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The lipase (E.C.3.1.1.3) used in the present study is the commercial Lipase (purity 99%, activity
103
16000 LU/g was purchased from Himedia Pvt. Ltd.The crude Hevea brasiliensis (rubber seed)
104
oil (Average molecular weight, 861.4 g mol-1, specific gravity, 0.919) was obtained from nearby
105
agricultural field in Thanjavur, India. Coal bottom ash is obtained from NLC India Limited
106
(Tamilnadu, India). Sodium hydroxide pellets (99%, Merck), ethanol (95%, Merck),
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hydrochloric acid (98%, Merck) are obtained from Merck Chemicals Pvt. Ltd. Methanol,
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dipotassium hydrogen phosphate, copper sulphate pentahydrate, lipase, sodium potassium
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tartarate, potassium dihydrogen phosphate, sodium carbonate, Folin‘sciocalteau reagent and gum
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arabic have been purchased from Himedia Laboratories Pvt. Ltd.
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2.1. Preparation of silica aerogel and Lipase immobilization
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Silica aerogel microspheres are prepared following the procedure reported in literature [22]. 0.1
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g of spherical aerogel particles was dispersed in 10 ml of potassium phosphate buffered to pH of
114
7. Precisely weighed lipase (10 mg) was added to the above mixture and stirred for 12 h,
115
maintaining temperature to 30 ºC. The immobilized lipase was separated by filtration and the
116
percentage immobilization and specific enzyme activity were determined. The enzyme
117
concentration was measured by Lowry method [23]. Specific enzyme activity for immobilised
118
enzyme was estimated by Olive oil emulsion method [24]. The percentage immobilization,
119
specific enzyme activity of the immobilized lipase on silica aerogel was 77% and 14800 U/ g.
120
2.2. Transesterification reaction
121
The transesterification reaction was carried out in a 100 mL screw capped vessel using 20 gm of
122
CHBO, 15 % (v/v %) water content, 430 mg immobilized lipase and at a constant temperature of
123
30˚C. Typically, higher water content ensures higher enzymatic activity [25]. However, the water
124
content was maintained at this level to: (1) prevent hydrolysis of the ester linkages at even higher
125
water content, thereby allowing for the forward transesterification reaction [26]; and (2) ensure
126
insignificant mass transfer interferences between the aqueous and oil phase (since enzyme lipase
127
have a unique feature to act at the interface between the aqueous and organic phases).
128
Methanol was added to the reaction vessel in a three step process at 1:8 molar ratio of
129
oil/methanol. For example, 2.72 ml of methanol was added to 22.5 mL of oil, which
130
corresponded to a molar ratio of 1:3. To increase the methanol: oil molar ratio to 1:9, 8.15 mL of
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methanol was added to 22.5 mL of oil, while keeping the amount catalyst at 430 mg. The
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reaction was carried out in a shaking incubator at 180 oscillations per minute. After a 10 h
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reaction, 100 ml of sample was taken from the reaction mixture and centrifuged. The upper layer
134
was analysed in GC-MS [27]. All the experiments were done in triplicates.
135
2.3 Response Surface Methodology (RSM)
136
Various experiments have been conducted to identify the important process variables, their effect
137
on the response variables and the interaction between the variables. Response Surface Method is
138
a statistical experimental design that helps identify optimum process conditions with less number
139
of experimental runs as compared to conventional experimental design. Thus, cost of expensive
140
experimental runs is minimized by RSM [28 - 29]. The main advantage in using RSM is the
141
speed and reliability of the outcome. Contours are response curves drawn in 2-dimensional
142
plane, keeping other variables fixed [30]. The shape of contour plots of the response helps us to
143
visualize interaction between the variables. A quadratic model was employed to express the
144
response variable in terms of the independent variables. Later the model was solved analytically
145
to obtain the optimum conditions [28]. All the statistical analyses were conducted using Minitab
146
statistics software (Version 16.2.2, Minitab Inc, Pennsylvania, USA). Analysis of variance
147
(ANOVA) was performed on the data to test the effects of the parameters and their interactions.
148
Tukey's multiple tests were performed to determine the differences among the levels of each
149
parameter. The α-level chosen was 0.05. The RSM utilized Taylor first order and second order
150
series with experimental data for optimization. The surface of Taylor expansion curve was
151
determined using RSM and this describes the response. It is of the form (Eqs.1):
152
Re sponse = β 0 + ∑ β i x i + ∑ β ii x 2 + ∑ β ij x i x j
153
Where,
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(1)
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βi, βij = Regression coefficients
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x= Process variable
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2.3 Catalyst and product characterization
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The synthesized catalyst matrix was characterized using Scanning Electron Microscope imaging
158
for surface morphology (6701 F, JEOL, Japan). Brunauer-Emmett-Teller (BET) method was
159
used to determine specific surface area of support matrix. Barrett-Joyner-Halenda model (BJH)
160
was used to determine pore size distribution. N2 gas adsorption technique was used to determine
161
volume of pores, average pore diameter and surface area of support matrixes. Methyl esters
162
formed during the experiment were examined by Gas Chromatography–Mass Spectroscopy
163
(CLARUS 500, PerkinElmer, USA). Fourier transform infrared spectroscopy (spectrum 100,
164
Perkin Elmer, USA) was used to characterize the functional group attached on mesoporous
165
silica. Quantification of methyl ester content in the reaction mixture was carried out using
166
(0.1mm x 10cm) gas chromatography capillary column. The column temperature was kept at 180
167
°C for 0.5 min, raised to 300 °C at 10 °C min-1 and maintained at this temperature for 10 min.
168
The temperatures of the injector and detector were set at 245 °C and 305 °C, respectively. The
169
fuel properties such as density, flash point, fire point, pour point, cloud point, kinematic
170
viscosity, Cetane number, Calorific value of biodiesel obtained from CHBO were measured
171
using ASTM methods.
172
3. RESULT AND DISCUSSION
173
3.1 Characterization
174
SEM image of silica aerogel (Figure 1) shows that the synthesized aerogel particles are in the
175
form of solid clusters with size ranging from 22 to 25 nm [31]. The BET surface area, average
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pore volume and average pore diameter were determined to be 443 m² g-1, 0.19 mL g-1 and 2.88
177
nm respectively (Supplementary Figure 1a & 1b). Figure 2 shows the FT-IR spectrum for the
178
bare and lipase immobilized silica aerogel. The presence of a broad band at about 3471 cm−1,
179
corresponding to the vibration of Si-OH terminal groups. An absorption peak at 1108 cm−1
180
corresponds to asymmetric stretching vibrations of siloxane bond Si–O–Si. The FT-IR spectrum
181
of silica aerogel after immobilization confirmed the presence of a number of functional groups in
182
the enzyme structure. Signal at 1661 and 1542 cm-1 generated by the N-H bending is the
183
characteristic for the pure enzymes corresponds to amide 1 and amide II bands [32]. Contribution
184
of the C–N stretching vibrations is more likely due to amide III (1426 cm−1) bands resulting from
185
N–H bending. Water is critical for enzymes structures, conformation and interaction with solid
186
hydrophilic surfaces. The 2961 cm-1 bands may be assigned to -OH bonded to the protein [33].
187
There was no change in spectrum frequency of surface functional group and disappearance of
188
any peak of silica aerogel, suggesting physical adsorption of the enzyme on the solid matrix.
189
3.2 Effect of oil-alcohol ratio on reaction (MR)
190
The rate of the reaction depends on the alcohol to oil mole ratio in the reaction mixture. If the
191
alcohol concentration is increased, an increased reaction rate can be observed. But too high
192
methanol concentration leads to deactivation of enzyme. Addition of alcohol in regular intervals
193
to maintain low concentration can improve the enzyme catalysis and will prevent the
194
denaturation. Figure 3 shows the variation of methanol to oil molar ratio on percentage yield of
195
biodiesel. The yield of biodiesel increases from 3:1 to 6:1 and reaches maximum yield of 93% at
196
8:1 and then decreases. Increasing trend in the biodiesel yield is due to higher concentration of
197
methanol and decreasing yield might be due to the denaturation of the immobilized lipase [34].
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199 3.3 Effect of Temperature (T)
201
Biodiesel production by enzymatic method requires milder conditions which is less energy
202
intensive when compared to the chemical method. It is well known that rates of most reactions
203
(enzymatic or chemical) tends to increase with temperature, and usually expressed by their
204
Arrhenius constant. In this study also, it was observed that the rate increased with temperature.
205
From the Figure 4, it was observed that the yield of biodiesel is highest (92%) at the 30°C [35].
206
However, most studies have limited their range of temperature investigated to below 40 °C for
207
two reasons: (1) Increasing temperature above 40 °C may denature the enzyme, decreasing the
208
rate of reaction; (2) good yields can be obtained at lower temperatures [36]. It was observed that
209
increase in temperature beyond 30°C caused the decline in the yield of biodiesel (Figure. 4).
210
Increasing the temperature from 20°C to 30°C showed a sudden rise in biodiesel yield owing to
211
higher reaction rate. As the temperature increases beyond 35°C the yield of biodiesel reduces due
212
to the loss of the enzyme activity [37]. It has been noted that lower temperature slows down the
213
rate, thereby prolonging the reaction time required to produce a similar yield. Increasing the
214
reaction temperature may lower yield due to the reversible nature of the reaction, and loss of
215
active sites due to enzyme denaturation [38].
216
3.1 Optimization parameters for biodiesel production
217
In this study, 3-level-3-factor design was implemented, totally 20 experiments were done in
218
duplicates [39]. The prophase research results showed that the three independent variable
219
parameters such as reaction temperature (T), molar ratios of methanol to oil (MR) and water
220
content (W) have important effects on FAME yield [33,34]. Water content (v/v) was 5-25%;
221
molar ratios of methanol to oil (mol/mol) was 3:1–8:1 and the reaction temperature was between
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30 and 40 (°C). The feedback was FAME yield in percentage (FAME). The non-dependent
223
factors, levels, and experimental model are tabulated in Table 2 and Table 3 [40, 41].
224
The model predicted was correlated to coefficients of interactions, linear and quadratic effects.
225
The correlation coefficients for each model and variable significance was measured by the
226
probability values are shown in Table 4.All the factors and their square interactions (P < 0.05)
227
except interaction term of methanol to oil molar ratio and water content were significant at the
228
The best fit for the experimental data (Table 2), expressed by polynomial model for the
229
percentage yield of FAME is as follows:
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− 0 .33 T 2 + 0 .202 M R2 − 0 .167 × W
2
− 0 .0159 T × W + 0 .000313 M R × W
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(2)
The R2 value determines the amount of variability in the observed response values that could be
232
described by the experimental factors and their interactions [41]. Adj-R2 value of 99.82% was
233
observed. As both R2 and Adj-R2 values are high and close to 1.0, a high link between the
234
observed and predicted values can be observed. This proves that the regression model provides
235
exceptional data on the relationship between independent variables and the response validation
236
of the model [42]. The optimum level of percentage yield of FAME was 94.33% at methanol to
237
oil molar ratio of 8:1, reaction temperature of 35ºC and 15% water content (v/v).
238
3.4 Reusability studies of immobilized lipase
239
The reusability of lipase immobilized on spherical silica aerogel was investigated by recovering
240
the immobilized particles after each reaction cycle (Figure 5). Approximately 90% of the activity
241
(in terms of methyl ester formation) was retained after 10 cycles of reaction. From the Figure. 5
242
it is evident that the gradual decrease in FAME yield was attributed to both loss of activity of
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immobilized lipase and loss of enzyme due to leaching. Lipase immobilized on silica aerogel can
244
be reused repeatedly without significant loss in activity in the production of biodiesel from
245
CHBO [43].
246
3.5 Fuel properties
247
Fuel properties of crude Hevea brasiliensis oil were determined by ASTM methods. Values of
248
flash point, fire point, pour point and cloud point were 149 ± 0.52°C, 191.6 ± 0.89°C, -4 ±
249
0.026°C, and -2 ± 0.10°C, respectively. Calorific value and Cetane number (38°C) of the
250
biodiesel produced were 37.9 (MJ/Kg) and 52 respectively. Kinematic viscosity and specific
251
gravity (38°C) of the biodiesel produced from rubber seed oil were 4.97 ± 0.35 (mm2/sec) and
252
0.831 ± 0.02 (g/cm3). Fuel properties of rubber seed Biodiesel were found to be compatible with
253
ASTM Biodiesel Standard (D 6751a) and European Biodiesel Standards (EN 14214). This
254
demonstrates the feasibility of crude Hevea brasiliensis oil biodiesel as fuel.
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257
In this work, we have demonstrated the production of biodiesel using Hevea brasiliensis, in an
258
immobilized lipase based transesterification process. The optimal condition for methanolysis was
259
8:1 molar ratio of methanol to oil and reaction temperature of 30°C. Under these conditions, 93
260
% yield of methyl ester were obtained. RSM based studies suggested a second order model, with
261
yield dependent on temperature, pH and methanol to oil molar ratio. The fuel produced from the
262
lipase based transestrification process was found compatible with ASTM Biodiesel Standard (D
263
6751a) and European Biodiesel Standards (EN 14214). Effective use of a waste material as a
264
support material for the enzymatic reaction helps reduce the production cost. The results suggests
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strongly that enzymatic transesterification of triglycerides offer an more environmentally
266
approach to biodiesel production, with potential for direct use of the product in diesel engines.
267
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dimension characterization of lipase B from immobilized onto titania at selected conditions,
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Magnetic Cellulose Nanocrystals. Sci. Rep . 6 (2016) 204-20.
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biodiesel production using transesterification process. Egypt. J. Petroleum. 25 (2016) 21-31.
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the art and prospective of lipase-catalyzed transesterification reaction for biodiesel production,
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High FFA Jatropha Curcas Oil Using Response Surface Methodology. Sci. 1 (2012) 36-43.
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Effect of water, organic solvent and adsorbent contents on production of biodiesel fuel from
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canola oil catalyzed by various lipases immobilized on epoxy-functionalized silica as low cost
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biocatalyst, J Mole Catal B: Enzym. 120 (2015) 93-99.
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water-resistant catalyst. Bioresource. Technol. 102 (2011) 2665-2671.
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6. List of figures and tables
417
Figure 1. SEM image of Silica aerogel.
418
Figure 2 FTIR spectra of bare and immobilized silica aerogel material.
419
19
ACCEPTED MANUSCRIPT
Figure 3: Effect of Methanol to oil molar ratio on immobilized lipase catalyzed methanolysis of
421
Hevea brasiliensis oil for Temperature 30ºC, 10% v/v water content and reaction time of 10 h.
422
Figure 4: Effect of temperature on immobilized lipase catalyzed methanolysis of Hevea
423
brasiliensis oil for 8: 1 molar ratio of methanol to oil, 10%v/v water content and reaction time of
424
10 h.
425
Figure 5: Immobilized lipase catalyzed methanolysis of Hevea brasiliensis oil for several cycles
426
at optimum conditions (8: 1 molar ratio of methanol to oil, 10%v/v water content, Temperature
427
30ºC and reaction time of 10 h).
428
Table 1: The comparison of biodiesel production from Hevea brasiliensis using various catalyst
429
in the literature with the present work.
430
Table 2: Experimental results based on central composite design.
431
Table 3: Estimated Regression Coefficients for yield of FAME
432 433
Table 4: Analysis of Variance for yield
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20
ACCEPTED MANUSCRIPT Table 1: The comparison of biodiesel production from Hevea brasiliensis using various catalyst in the literature with the present work.
Operating parameter’s
Lipase from Aspergillus niger
8:1 molar ratio of methanol to oil, 15 % water content and 35°C, 430 mg immobilized lipase/g oil, reaction time 7 h. Methanol to oil molar ratio of 3:1– 15:1, a catalyst concentration of 0.25– .25%wt., a reaction temperature of 50–70 °C, a reaction time of 30–150 min and speed agitation of 800–1200 rpm
93
SC
Acid esterification: KOH
% yield
Work
RI PT
Catalyst
Present work
A.S. Silitonga et al, 2016 [13]
31
Oryzae Lipase
Methanol to oil molar ratio of 1:4 and catalyst concentration of 15(w/w) % of oil after 48 h.
V.V. Vipin et al, 2016 [52]
Activated carbon impregnated with pure KOH
Reaction temperature 55 °C, Reaction time 60 min, catalyst loading 3.5 wt% and methanol to oil ratio 15:1.
89.81
Sumit H. Dhawane et al, 2015 [14]
84
S. Karnjanakom et al, 2015 [15]
Temperature 60 °C, reaction time 1 h, and 5 g of carbon based catalyst at varying quantities of catalyst loading (0.5, 2, 3.5, 5 wt%) and methanol to oil ratio (5:1–20:1)
89.3
Sumit H. Dhawane et al, 2015 [16]
6:1 alcohol to oil ratio, 1% catalyst concentration, 55 °C reaction temperature and 67.5 min reaction time.
96.8
Junaid Ahmad et al, 2014 [20]
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SO3H-MCM-41
Methanol to oil ratio 16:1,Catalyst loading of 14.5 wt.% and a reaction time and temperature of 48 h and 129.6°C,
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Rhizopus
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Base transesterification: H2SO4
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99.32
Carbon based KOH impregnated heterogeneous catalyst from flamboyant pods (Delonix regia) Acid esterification: H2SO4
Base transesterification: KOH
ACCEPTED MANUSCRIPT Catalyst concentration in range 0.11%(v/v), raw material to methanol molar ratio (1:2) , Temperature 60 °C , ratio of raw material to methanol in range 1:1.5-1:3 and reaction time 60 min. Catalyst loading of 5 wt. %; methanol to oil molar ratio of 4:1; reaction temperature of 65°C and reaction time of 4 h
Alkaline esterification: NaOH Base transesterification: NaOH Alkaline esterification: H2SO4
NaOH
96.9
SC
Methanol/oil molar ratio of 9:1 and 0.5% by weight of sodium hydroxide
1% catalyst loading, 6:1 alcohol to oil ratio, 97.8 ± 0.2 C °C reaction temperature and 60 min reaction time.
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NaOH
Methanol/oil ratio 0.28 % v/v, sodium hydroxide of 0.75% w/v, Temperature 51.23 °C, Reaction time 82.52 min
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Base transesterification:
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Poly (sodium acrylate) supported NaOH
Methanol/oil molar ratio 6:1, stirring speed 1000 rpm, reaction temperature 60 °C, 3 wt% NaOH/NaPAA sample with NaOH loading amount of 7.5 mmol/g was used as catalyst
TE D
Limestone based catalyst
91.05
Widayat et al, 2013 [19]
RI PT
H2SO4
Jolius Gimbun et al, 2013[47]
96
Ru Yang et al, 2013 [48]
97.1
D.F. Melvin Jose et al, 2013 [49]
-
A.S. Ramadhas et al, 2005 [50]
84
O.E. Ikwuagwu et al, 2000 [51]
ACCEPTED MANUSCRIPT Table 2: Experimental results based on central composite design. Pred
Blocks
T
MR
W
% yield
% yield
1
1
1
30
4
5
66.30
66.40
2
2
1
1
40
4
5
59.30
59.18
3
3
1
1
30
8
5
69.10
68.99
4
4
1
1
40
8
5
71.20
71.07
5
5
1
1
30
4
25
64.67
64.82
6
6
1
1
40
7
7
1
1
30
8
8
1
1
9
9
-1
1
10
10
-1
1
11
11
-1
1
12
12
-1
13
13
-1
14
14
-1
15
15
16
RI PT
1
SC
StdOrder RunOrder PtType
Exp
25
54.28
54.41
8
25
67.30
67.44
40
8
25
66.40
66.33
30
6
15
83.21
82.82
40
6
15
78.61
78.65
35
4
15
86.55
86.19
TE D
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4
35
8
15
93.44
93.44
1
35
6
5
73.76
73.88
1
35
6
25
71.20
70.72
0
1
35
6
15
89.20
89.01
16
0
1
35
6
15
88.45
89.01
17
0
1
35
6
15
89.40
89.01
18
0
1
35
6
15
88.12
89.01
19
19
0
1
35
6
15
89.77
89.01
20
20
0
1
35
6
15
88.66
89.01
18
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1
ACCEPTED MANUSCRIPT Table 3: Estimated Regression Coefficients for yield of FAME
Term
Coef
SE
T
P
Coef 89.0349
0.1763
505.067
0.000
T
-2.0792
0.1622
-12.822
0.000
MR
3.6340
0.1622
22.410
0.000
W
-1.5810
0.1622
-9.750
0.000
T*T
-8.2784 0.8076
T*W
SC
0.000
0.3092
2.612
0.026
0.3092
-54.030
0.000
0.1813
12.817
0.000
-0.7988
0.1813
-4.406
0.001
0.0062
0.1813
0.034
0.973
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MR *W
-26.772
2.3238
EP
T* MR
16.7074
0.3092
TE D
W*W
M AN U
MR * MR
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Constant
S = 0.512786 R-Sq = 99.90%
PRESS = 7.25860 R-Sq(pred) = 99.73%
R-Sq(adj) = 99.82%
ACCEPTED MANUSCRIPT Table 4: Analysis of Variance for yield
Source
Seq SS
Adj SS
Adj MS
F
Regression
9
2698.97
2698.97
299.885
1140.47
0.000
Linear
3
200.29
200.29
66.762
253.90
0.000
T
1
43.23
43.23
43.231
164.41
0.000
MR
1
132.06
132.06
132.060
502.22
0.000
W
1
25.00
25.00
24.996
95.06
0.000
Square
3
2450.38
2450.38
816.793
3106.28
0.000
T*T
1
1587.44
188.46
188.461
716.72
0.000
MR * MR
1
95.31
1.79
1.794
6.82
0.026
W*W
1
767.62
767.62
767.624
2919.29
0.000
Interaction
3
48.30
48.30
16.101
61.23
0.000
T* MR
1
43.20
43.20
43.199
164.28
0.000
T*W
1
5.10
5.10
5.104
19.41
0.001
MR *W
1
0.00
0.00
0.000
0.00
0.973
0.34
0.868
Lack-of-Fit
Total
SC
M AN U
TE D 2.63
2.63
0.67
0.67
0.134
5
1.96
1.96
0.392
19
2701.60
P
0.263
5
AC C
Pure Error
10
EP
Residual Error
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DF
EP
TE D
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RI PT
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Figure 1. SEM image of Silica aerogel.
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Figure 2 FTIR spectra of bare and lipase immobilized silica aerogel material.
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100
RI PT SC
60
40
M AN U
% yield of biodiesel
80
20
0 4
6
TE D
2
8
10
12
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Methanol to oil molar ratio
Figure 3: Effect of Methanol to oil molar ratio on immobilized lipase catalyzed methanolysis of
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Hevea brasiliensis oil for Temperature 30ºC, 10% v/v water content and reaction time of 10 h.
ACCEPTED MANUSCRIPT
100
RI PT SC
60
40
20
0 20
25
30
35
40
TE D
15
M AN U
% Yield of biodiesel
80
Temperature (°C)
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Figure 4: Effect of temperature on immobilized lipase catalyzed methanolysis of Hevea
10 h.
AC C
brasiliensis oil for 8: 1 molar ratio of methanol to oil, 10%v/v water content and reaction time of
ACCEPTED MANUSCRIPT
100
RI PT SC
60
40
20
0 1
2
3
4
5
6
7
8
9
10
11
TE D
0
M AN U
% Yield of biodiesel
80
EP
Number of cycles
Figure 5: Immobilized lipase catalyzed methanolysis of Hevea brasiliensis oil for several cycles
AC C
at optimum conditions (8: 1 molar ratio of methanol to oil, 10%v/v water content, Temperature 30ºC and reaction time of 10 h). .
ACCEPTED MANUSCRIPT
Research highlight •
Biodiesel was produced from Hevea brasiliensis by lipase immobilized on a low cost
•
Ordered mesoporous spherical silica aerogel could be secured from cheaper silica precursor originating from coal bottom ash.
The immobilized enzyme, when reused, showed highly effective methanolysis for ten
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•
cycles.
Use of immobilized silica aerogel for biodiesel production was more green and cost
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•
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support.
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effective.