Enhanced Enzymatic Transesterification of Palm Oil to Biodiesel

Accepted ManuscriptTitle:Enhanced Enzymatic Transesterification of Palm Oil toBiodieselAuthors:Md.Mahabubur Rahman Talukder,Probir Das,TanShu Fang,Jin Chuan WuPII:S1369-703X(11)00083-0DOI:doi:10.1016/j.bej.2011.03.013Reference:BEJ5307To appear in:Biochemical Engineering JournalReceived date:29-10-2010Revised date:26-2-2011Accepted date:28-3-2011Please cite this article as:Md.M.R.Talukder,P.Das,T.S.Fang,J.C.Wu,Enhanced Enzymatic Transesterification of Palm Oil to Biodiesel,Biochemical Engineering Journal(2010),doi:10.1016/j.bej.2011.03.013This is a PDFfile 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 itsfinal 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.A cc ep te dMa nu sc ri p t1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Enhanced Enzymatic Transesterification of Palm Oil to BiodieselMd. Mahabubur Rahman Talukder*, Probir Das, Tan Shu Fang, Jin Chuan Wu,Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, Singapore 627833, Singapore* Corresponding author. Tel.: +65 67963826; Fax: +65 63166182. E-mail address: talukder@.sg*revised manuscriptA cc ep te dMa nu sc ri p t4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ABSTRACTInefficient reaction is one of the drawbacks for the enzymatic production of biodiesel (BD). A method for enhanced Novozym 435-catalyzed transesterification ofpalm oil (PO) to BD using a mixture of methanol and methyl acetate (MA) as acylacceptors has been developed. BD yield using methanol-MA mixture reached 95-96% after8 h at a methanol to PO molar ratio of 1:1, a MA to palm oil molar ratio of 12:1, 2 g of PO,temperature of 50 ˚C and Novozym 435 loading of 0.6 g. In contrast with methanol-MA mixture, BD yield using MA alone after 24 h was 88-89 % under the same reactionconditions. Novozym 435 was repeatedly used at 40 and 50 ˚C for 10 cycles. There was noconsiderable loss in the enzyme activity at 40 ˚C using either methanol-MA mixture orMA alone, while the remaining activity of the enzyme at 50 ˚C in both cases after 10cycles was 82-85 %. The developed method has a potential to be used for efficient enzymatic production of BD.Keywords : biodiesel, Novozym 435, methanol-methyl acetate mixture, palm oil, transesterificationA cc ep te dMa nu sc ri p t4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1. IntroductionNovozym 435-catalyzed transesterification of vegetable oil with methanol has been extensively reported for the production of BD, although this technology has not receivedmuch commercial attention except in China, where the first industrial scale for BDproduction in the world (with lipase as the catalyst at a capacity of 20,000 tons year -1) is inoperation [1]. The major limitations of using lipase (e.g. Novozym 435) in biodieselproduction include: (1) longer reaction time, (2) methanol deactivation of lipase due to low solubility of methanol in vegetable oil (3) glycerol inhibition due to its adsorption onlipase. Different techniques have been reported to minimize such limitations. Organicsolvents with large molar excess of methanol have been used [2-4]. A very lowconcentration of methanol during the reaction was maintained by stepwise addition ofmethanol [5, 6]. Salts saturated solutions [7] and silica-gel [8] based controlled release system for methanol have also been applied. However, these techniques have some inherent problems. The removal of organic solvents as well as excess methanol is not economically favorable. Maintaining methanol concentration at a very low level bystepwise addition cannot be an appropriate approach for the large scale production of BD.The presence of salt saturated solution or silica-gel causes difficulties in downstream separation. Recently, MA instead of methanol has been used as an acyl acceptor [9, 10]. However, the reaction with MA still needs longer time.In this study, enhancement in Novozym 435-catalyzed transesterification of PO to BD using a mixture of methanol and MA as acyl acceptors was attempted. The results obtained using methanol-MA mixture and MA alone were compared. The presence of MA minimized the methanol deactivation of lipase, and the combined effect of methanol andA cc ep te dMa nu sc ri p t4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 MA leads to enhance transesterification of PO to BD as methanol is more active than MA [10].2. Materials and methods2.1 MaterialsNovozym 435 (Candida antarctica lipase B immobilized on acrylic resin), PO, MAand standard methyl esters (98-99%) were from Sigma. The saponification value of POwas 200 mg KOH/g, which was determined by a method reported previously [11]. The average molecular weight of palm oil is 840. The major fatty acids component of palm oilare palmitic acid (35-48%), oleic acid (35-50%), linoleic (6-13%), stearic (3-7%) andmyristic acid (0.5-6%). HPLC grade methanol, isopropanol and hexane were from J.T.Baker, USA. All chemicals unless mentioned otherwise were of analytical grade and usedas received.2.2. Transesterification of palm oil with MA alone and MA-methanol mixture2 g of palm oil and methanol-MA mixture or MA alone were mixed in a 25 ml screw caped glass bottle for 10 min at 250 rpm and 50 ˚C by shaker incubator. Thereaction was initiated by adding Novozym 435. Novozym 435 amount, molar ratio of MAto PO and methanol to PO were varied as mentioned in the respective Figures. 2.3 HPLC analysis of BDAfter the specified reaction time, the sample (0.5 ml) was mixed with hexane (4.5ml), and centrifuged at 4000 rpm and 25˚C for 10 min . The BD concentration in upper layer was determined by HPLC [12, 13]. HPLC (Waters 2695, USA) was equipped with a UV detector (Waters 2487, USA) and a prevail-C18 5u column (4.6 mm 250 mm, Altech Inc., USA). The UV wavelength and the column temperature were set at 210 nm and 40˚C ,A cc ep te dMa nu sc ri p t4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 respectively. The mobile phase consisted of three different components: hexane, isopropanol and methanol. Reservoir A contained methanol and reservoir B contained a mixture of isopropanol and hexane (5:4, v/v). The gradient went from 100% A to 50% A +50% B linearly over 30 min. The flow rate of the mobile phase was 1 ml/min and thesample injection volume was 10 l. BD was quantified according to the externalcalibration curves. Methyl oleate, methyl palmitate, methyl stearate, methyl linoleate andmethyl myristate were used as standards for BD because oleic, palmitic, stearic, linoleicand myristic acids are major fatty acids in PO. BD yield was defined as the amount of BDproduced per gram of PO. A typical (e.g. at biodiesel yield of 95%), the compositions ofpalmitic, oleic, linoleic, stearic and myristic esters in biodiesel are 42, 43, 7, 5 and 4%,respectively. The range of palmitic, oleic, linoleic, stearic and myristic acid esters in biodieselvaried in the range of 42-44%, 43-45%, 4-7%, 4-5% and 2-4%, respectively.3. Results and discussion3.1 Comparison of MA and methanol as acyl acceptorBD yield after 24 h with different MA to PO molar ratio was shown in Figure 1.BD yield increased with the increase in MA concentration up to MA to PO ratio of 8:1,after which it remained almost the same even at a ratio of 12:1. This result indicates thatthe reaction is saturated with MA at a ratio of ≥ 8:1. Hence, further increase in MAconcentration cannot improve the reaction. The similar result also reported by Du et al 10. In contrast with MA, the presence of more than 1/3 stoichiometric amount of methanol deactivates Novozym 435 and the transesterification of PO is completely ceased at methanol to PO molar ratio of 3:1 [14]. Three successive additions of 1/3 stoichiometric amount of methanol was, therefore, adopted and BD yield of 95% was obtained after 30 h (Figure 2). While BD yield with MA reached only about 70% after 30 h regardless of MAA cc ep te dMa nu sc ri p t4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 to PO ratio of 8:1 or 12:1. These results indicate that reaction between triglycerides and methanol (i.e. transesterification) progressed faster than that between triglycerides and MA (i.e. ester exchange). This could be due to the higher reactivity of methanol compared toMA. MA is a bifunctional group-donor, and can be used as a source of both methanol fortransesterification and acetic acid for acetylation. Because of this bifunctional characteristic of MA,the equilibrium BD yield with MA could be lower than that with methanol. In order to improve theyield and reaction efficiency, the combined use of methanol and MA was attempted. Since thelimited miscibility of methanol with PO causes the inactivation of Novozym 435 and MAhelps the miscibility, an excess amount of MA (i.e. MA to PO ratio of 12:1) was used inthe subsequent studies.3.2 Transesterification of PO with methanol-MA mixtureThe transesterification of palm oil with methanol-MA mixture at different methanolto PO molar ratios but at a fixed MA to PO ratio of 12:1 was shown in Figure 3. Thereaction with methanol-MA mixture progressed much faster than that with MA alone. The increase in methanol to PO molar ratio fastened the reaction although a slight inhibitionwas noticed at a ratio of 3:1. This inhibition might be attributed to the formation ofbyproduct glycerol, which adsorbs onto Novozym 435, and reduce the substrate diffusion.The higher BD yield with methanol-MA mixture even at methanol to PO molar ratio of 3:1suggests that the presence of MA minimized the methanol deactivation of Novozym 435. Since higher methanol concentration may lead to the formation of glycerol, methanol to PO ratio of 1:1 was chosen for the subsequent studies. At low methanol to palm oil molar ratioof ≤1:1, byproduct could be a mixture of diacetylglycerol and triacetylglycerol. The possibility of formation of monoacetylglycerol is very low due to the presence of excess MA (12:1). Furthermore, monoacetylglycerol and diacetylglycerol can be converted into triacetylglycerol byA cc ep te dMa nu sc ri p t4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 reacting with MA. Triacetylglycerol can be used as biodiesel additive, and is not necessary to remove from biodiesel.3.3 Effect of Novozym 435 loadingThe time course of the transesterification of palm oil with methanol-MA mixtureand MA alone at different Novozym 435 loading were shown in Figure 4a and 4b,respectively. The rate of reaction increased with the increase in Novozym 435 loading andreached a maximum level at 20 and 30 wt% of substrate (PO) for methanol-MA mixture and MA alone, respectively. Du et al. [10] have found that the rate of reaction with MA islower than that with methanol, consequently the more lipase used. Hence, the use ofmethanol-MA mixture reduced the amount of Novozym 435 required (20 wt%) for themaximum reaction rate. The maximum BD yield with methanol-MA mixture and MAalone was 95-96% after 8 hr (Figure 4a) and 88-89% after 24 h (Figure 4b), respectively.The transesterification of PO to BD with methanol-MA mixture is, therefore, at least 3-foldefficient than that with MA alone. 3.4 Novozym 435 recyclingThe Novozym 435 recycling with methanol-MA mixture and MA alone wasperformed at 40 and 50 ˚C . Each cycle was set at 7 and 24 h for methanol-MA mixture andMA alone, respectively. After each cycle, Novozym 435 was recovered by filtration(Whatman 125), washed with 5-10 ml of MA and subsequently reused. The remaining activity of the enzyme was defined as percentage of BD yield at a given cycle compared to the yield at 1st cycle.Figure 5 shows that Novozym 435 could be repeatedly used at 40 ˚C over 10 cycles without considerable loss of its activity regardless of using methanol-MA mixture (Figure 5a) or MA alone (Figure 5b). However, the enzyme activity slightly dropped during theA cc ep te dMa nu sc ri p t4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 repeated use at 50 ˚C although the maximum rate of reaction was found at 50-60 ˚C in this study and elsewhere [7]. The remaining activity of Novozym 435 after 10 cycles at 50 ˚C was 82-85% for both methanol-MA mixture (Figure 5a) and MA alone (Figure 5b). Thisresult suggests that the operating temperature for prolong use of Novozym 435 should bekept at around 40˚C to avoid heat induced deactivation. Du et al. [10] have reported thatNovozym 435 can be repeatedly used at 40 ˚C over 100 cycles when MA is used as acylacceptor. 4. ConclusionsAn improved and novel approach for Novozym 435-catalyzed transesterification ofPO with methanol-MA mixture as acyl acceptor was investigated. Methanol is more activethan MA but it deactivates Novozym 435 at methanol to PO molar ratio of >1:1. Methanoldeactivation of Novozym 435 was minimized by the presence of MA in the reaction mixture, and methanol to PO molar ratio of 1:1 was found to be the most effective for enhanced transesterification of PO to BD when MA to PO molar ratio was 12:1. The reaction with methanol-MA mixture progressed 3-fold faster than that with MA alone.Novozym 435 can satisfactorily be recycled either using methanol-MA mixture or MAalone. This novel approach of using methanol-MA mixture as acyl acceptor provides an improved method for enzymatic production of BD from vegetable oils.AcknowledgementFinancial support from the Agency for Science Technology and Research (A*STAR) of Singapore is gratefully acknowledged.A cc ep te dMa nu sc ri p t4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Figure CaptionsFig. 1 Effect of MA to PO molar ratio on BD yield after 24 h of reaction. Reaction conditions: 2 g of PO, 0.1 g of Novozym 435 (5 wt% of PO), temperature of 50 °C andshaking speed of 250 rpm.Fig. 2 Comparison of methanol and MA as acyl acceptor for transesterification of PO toBD. Reaction conditions: 2 g of PO, 0.1 g of Novozym 435, temperature of 50 °C andshaking speed of 250 rpm. Methanol (1/3 stoichiometric amount) was added three times at 0, 4 and 16 h of reaction.Fig. 3 Transesterification of PO to BD with methanol-MA mixture at different methanol toPO molar ratios. Reaction conditions: 2 g of PO, 0.1 g of Novozym 435, MA to PO molarratio of 12:1, temperature of 50 °C and shaking speed of 250 rpm.Fig. 4 Effect of Novozym 435 loading on transesterification of PO to BD using methanol-MA mixture (a) and MA alone (b). Reaction conditions: 2 g of PO, temperature of 50 °C, MA to PO molar ratio of 12:1, methanol to PO molar ratio of 1:1 and shaking speed of 250 rpm.Fig. 5 Novozym 435 recycling at reaction temperature of 40 and 50 °C. Each cycle was setfor 7 and 24 h for methanol-MA mixture (a) and MA alone (b), respectively. Reaction conditions: 2 g of PO, MA to PO molar ratio of 12:1, methanol to PO molar ratio of 1:1, 0.4 g of Novozym 435 for methanol-MA mixture, 0.6 g for MA alone and shaking speed 250 rpm. The method of recycling was described in the text (section 3.4).A cc e p t ed Ma nu sc ri p t4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 601020304050607034681012MA to PO molar ratioB D y i e l d (%)Fig.1A cc e p t ed Ma nu sc ri p t5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 0102030405060708090100061218243036Time (h)B D y i e l d (%)3-step addition of methanolMA to PO ratio 8:1MA to PO ratio 12:1Fig. 2A cc ep te d5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 0204060801000481216202428Time (h)B D y i e l d (%)Fig. 3A cc ep te dM a nu sc ri p t5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 02040608010024681012Time (h)B D y i e l d (%)5 wt%of palm oil10 wt% of palm oil20 wt% of palm oil 30 wt% of palm oil20406080100061218243036Time (h)B D y i e l d (%)5 wt% of palm oil 10 wt% of palm oil 20 wt% of palm oil 30 wt% of palm oil 40 wt% of palm oilFig. 4(a)(b)A cc ep te dMa nu sc ri p t5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 02040608010012345678910Cycle No.R e m a i n i n g A c t i v i t y (%)40 °C50 °C2040608010012345678910Cycle No.R e m a i n i n g A c t i v i t y (%)40 °C 50 °CFig. 5(a)(b)A cc ep te dMa nu sc ri p t4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 References[1] W. 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