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Responsesurfacemethodologyassistedbiodieselproductionfromwastecookingoilusingencapsulatedmixedenzyme

ARTICLEinWASTEMANAGEMENT·AUGUST2015

ImpactFactor:3.16·DOI:10.1016/j.wasman.2015.07.036·Source:PubMed

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SirajunnisaAbdulRazack

AnnamalaiUniversity

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SurendhiranDuraiarasan

AnnamalaiUniversity

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Availablefrom:SirajunnisaAbdulRazack

Retrievedon:06September2015

Waste Management xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Waste Management

journal homepage: www.elsevier .com/locate /wasman

Response surface methodology assisted biodiesel production fromwaste cooking oil using encapsulated mixed enzyme

http://dx.doi.org/10.1016/j.wasman.2015.07.0360956-053X/� 2015 Elsevier Ltd. All rights reserved.

E-mail addresses: siraj.razack@gmail.com (S.A. Razack), suren_micro@yahoo.co.in (S. Duraiarasan)

Please cite this article in press as: Razack, S.A., Duraiarasan, S. Response surface methodology assisted biodiesel production from waste cooking oencapsulated mixed enzyme. Waste Management (2015), http://dx.doi.org/10.1016/j.wasman.2015.07.036

Sirajunnisa Abdul Razack, Surendhiran DuraiarasanBioprocess Laboratory, Department of Chemical Engineering, Annamalai University, Annamalainagar, Tamil Nadu 608002, India

a r t i c l e i n f o

Article history:Received 20 March 2015Revised 29 June 2015Accepted 20 July 2015Available online xxxx

Keywords:Waste cooking oilInteresterificationLipaseBiodieselResponse surface methodology

a b s t r a c t

In the recent scenario, consumption of petroleum fuels has increased to greater height which has led todeforestation and decline in fossil fuels. In order to tackle the perilous situation, alternative fuel has to begenerated. Biofuels play a vital role in substituting the diesel fuels as they are renewable and ecofriendly.Biodiesel, often referred to as green fuel, could be a potential replacement as it could be synthesized fromvaried substrates, advantageous being the microalgae in several ways. The present investigation wasdealt with the interesterification of waste cooking oil using immobilised lipase from mixed culturesfor biodiesel production. In order to standardize the production for a scale up process, the parametersnecessary for interesterification had been optimized using the statistical tool, Central CompositeDesign – Response Surface Methodology. The optimal conditions required to generate biodiesel were2 g enzyme load, 1:12 oil to methyl acetate ratio, 60 h reaction time and 35 �C temperature, yielding amaximum of 93.61% biodiesel. The immobilised lipase beads remain stable without any changes in theirfunction and structure even after 20 cycles which made this study, less cost intensive. In conclusion, thestudy revealed that the cooking oil, a residue of many dining centers, left as waste product, can be used asa potential raw material for the production of ecofriendly and cost effective biofuel, the biodiesel.

� 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Burgeoning population, surging globalization, soaring energyneeds, rapid rise in oil prices and diminishing fossil fuels haveurged on the biotechnologists to explore alternate fuels. To fulfillthe incorrigible demand of petrofuels, biodiesel has beenascertained the most probable substitute. Biodiesel, generallyreferred to as fatty acid methyl esters (FAME), is a renewable,non toxic, sulfur-less, free from carbon dioxide and a clean fuel(Arumugam and Ponnusami, 2014; Leung et al., 2010). This couldtypically, be produced using vegetable oils, animal fats as feed-stocks, that determine the generation of biofuels, through esterify-ing reactions aided by catalysts. Vegetable oils are potentialsubstrates for yielding biodiesel since they are natural, renewableand ecofriendly but requirements of land for larger crop cultiva-tion, deforestation and their high market values make them insa-tiable for larger biodiesel generation (Leung et al., 2010); freshvegetable oils are also high-priced when compared to fossil fuels(Chhetri et al., 2008). Feedstock and biodiesel majorly account for80% of the overall economy of the process (Chhetri et al., 2008;Kuan et al., 2013). Hence, waste cooking oil (WCO) could be a

promising and cost effective candidate for synthesizing the biofuel,due to its low cost and availability elsewhere (Omar and Amin,2011; Gnanaprakasam et al., 2013; Yan et al., 2014).

Commercially, biodiesel production is carried out by acid oralkali mediated transesterification of substrates to FAME. Thoughthese conventional methods yield satisfactorily and require lowreaction time, they possess their own disadvantages like difficultrecovery of glycerol, low conversion rate, high-energy require-ment, saponification and necessity of wastewater treatment(Rahimi et al., 2014; Jiang et al., 2014; Cervero et al., 2014; Leeet al., 2013; Rodrigues and Ayub, 2011; Kawakami et al., 2011).To overcome these difficulties, enzymatic mode has become agreen and substantial means of biodiesel synthesis. Through thistechnique, easy recovery of by-product, elimination of salts andcatalyst, higher yield under normal conditions and low tempera-ture could be highly achieved (Lee et al., 2013; Gumbyte et al.,2011; Gharat and Rathod, 2013). Enzymatic biodiesel productionhas the ability to utilize low quality feedstocks like WCO with highFFA, which is another important criterion to perform using thismethod than that of the conventional (Kuan et al., 2013).

Lipase is one of the foremost and efficient enzymes imple-mented in enzymatic conversion of oil to FAME. Lipases could beutilized for the production since there is no production of soap,can be performed under milder conditions, ecofriendly and FAME

il using

2 S.A. Razack, S. Duraiarasan / Waste Management xxx (2015) xxx–xxx

purification is easier than in conventional techniques (Zarei et al.,2014; Adachi et al., 2013). Lipases could be easily activated inthe presence of oil water interface and has the capacity to maintainthe catalytic activity in non-aqueous, biphasic systems andmicellar solutions (Antczak et al., 2009).

However, biocatalytic esterification method has its own certainobstrucle like high cost of enzyme and slow reaction (Arumugamand Ponnusami, 2014). Recyclability of lipases is another majorphenomenon that has to be considered essentially, which couldbe accomplished by immobilization. Various techniques to immo-bilise lipases had extensively been studied earlier (Modi et al.,2007; Kuan et al., 2013). In accordance with the report by Kuanet al. (2013), entrapment and encapsulation of the enzymes aremore advantageous than any other immobilization method dueto the prevalence of high mass transfer resistance.

Transesterification of oils to biodiesel is carried out in the pres-ence of short chain alcohols such as methanol and ethanol. Duringthe process, the prime by-product formed is glycerol, hindering thecatalytic reactions and its downstream processing (Modi et al.,2007). Thus, interesterification using methyl acetate could be abetter alternative due to fast reaction rates, high biodiesel yieldand by-product being triacetin (triacetylglycerol), which does notdeactivate lipase unlike glycerol. Moreover, triacetin is used com-mercially in polymers and explosives as gelatinizing agents andin tobacco, pharmaceutical and cosmetic industries as additives(Campanelli et al., 2010; Casas et al., 2011; Maddikeri et al.,2013). Moreover many researches are focused on finding optimallipases to catalyze the biodiesel production. But natural oils arenot homogenous substrates, as they are comprised of triglycerideswith different fatty acids. Additionally, reaction mixture consists oftriglycerides and free fatty acids, hence enzyme should own theability to catalyze varied substrates; these make the finding of anoptimal lipase tedious. Hence a mixture of different lipases withspecific characters that act upon several substrates could act asan optimal biocatalyst (Rodrigues and Ayub, 2011; Garcia et al.,2011).

The two strains namely Bacillus subtilis and Burkholderia cepaciaproduce alkaline thermotolerant lipase which work at higher tem-perature in the ranges between 50 �C and 70 �C, more tolerant toorganic solvents (Li and Yan, 2010; Sivaramakrishnan andMuthukumar, 2012) and can perform at high pH level (Dalalet al., 2008). Hence these two strains were selected to producelipase for biodiesel synthesis. The main objective of the study ofthe present investigation is the enzymatic interesterification ofWCO to biodiesel using mixed lipase from B. subtilis and B. cepacia(MTCC 1617), with methyl acetate as an acyl acceptor and optimiz-ing statistically by Response Surface Methodology (RSM), based on2 level 4 factor Central Composite Design (24 factorial design).These experiments let us learn the effect of each independent fac-tor studied – biocatalyst loading, molar ratio, temperature andreaction time, and the interactive effects between the parameterson the dependent response variable. Central Composite Design(CCD) has the vantage of predicting the responses within the limitsby varying the parameters simultaneously.

2. Materials and methods

2.1. Sample collection and pretreatment of WCO

Waste cooking oil (WCO) was purchased from local restaurantnear Annamalai University campus, Annamalai Nagar. The WCOsample was filtered using a filter cloth to separate fried wastes inthe oil. Then the filtered oil was washed with water to removewater soluble salts present in the oil sample. After that the WCOwas heated at 110 �C in a beaker to evaporate the excess water.

Please cite this article in press as: Razack, S.A., Duraiarasan, S. Response surfacencapsulated mixed enzyme. Waste Management (2015), http://dx.doi.org/10.

The saponification (SV) and acid value (AV) were analyzed fordetermining molecular weight of WCO (Sathasivam andManickam, 1996; Xu et al., 2006). The molecular weight of wastecooking oil was found to be 855.68 g from the saponification valueof 196.3 mg KOH g�1 and acid value of 1.68 mg KOH g�1. The fattyacid compositions of WCO are palmitic acid (C16:0) – 6.73%, stearicacid (C18:0) – 4.11%, oleic acid (C18:1) 36.54% and linoleic acid(C18:2) – 52.62%.

2.2. Bacterial strains and culture medium

Two bacterial strains were used for lipase mediated biodieselproduction (Fig. 1). B. cepacia (MTCC 1617) and B. subtilis wereobtained from Department of Microbiology, AnnamalaiUniversity, Tamilnadu, India. The two bacterial cultures werecultivated using 100 ml of two different nutrient broth consist ofpeptone (1.0), yeast extract (0.3), beef extract (0.3) and NaCl(0.3) (g/100 ml) and incubated at 37 �C for 24 h. These cultureswere used as seed culture for lipase production.

2.3. Fermentation for lipase production

The lipase production was carried out in two different 250 mlErlenmeyer flask using 100 ml basal medium containing 2% oliveoil, 0.2% CaCl2�2H2O, 0.01% MgSO4�7H20, 0.04% FeCl3�6H2O and0.3% NaCl. The contents were incubated for 48 h at 37 �C at200 rpm. The pH was maintained at 7. After incubation, thecultures were centrifuge at 10,000 rpm for 10 min at 4 �C. Thecrude lipase (culture supernatant) from two bacterial cultureswere mixed together and partially purified by 70% ammoniumsulfate and kept at 4 �C for overnight. Then the precipitate was col-lected by centrifugation at 10,000 rpm for 30 min at 4 �C and it wasused for immobilization.

2.4. Lipase assay and immobilization of partially purified lipase byencapsulation

Lipase activity was determined according to Burkert et al.(2004) and Padilha et al. (2012). The immobilization was carriedby the encapsulation method using 2% sodium alginate. The par-tially purified lipase mixer was added with sodium alginate solu-tion. The mixer was dripped into cold sterile 0.2 M CaCl2 usingsterile syringe from a constant distance and was cured at 4 �C for1 h. The beads were hardened by suspending it again in a freshCaCl2 solution for 24 h at 4 �C with gentle agitation. After immobi-lization, the beads were separated by filtration and washed with25 mM phosphate buffer (pH 6.0), to remove excess calcium chlo-ride and enzyme.

2.5. Optimization of enzymatic biodiesel production using RSM

The production of biodiesel from WCO was developed and opti-mized using response surface methodology (RSM) provided byDesign-Expert software version 8.0.7.1 (Stat-Ease Inc.,Minneapolis, USA). A standard RSM design tool known as CentralComposite Design (CCD) was applied to study the catalyzed inter-esterification reaction variables. These experiments help in learn-ing the effect of each independent factor and the interactiveeffects between the parameters on the dependent response vari-able. Central Composite Design (CCD) has the vantage of predictingthe responses within the limits by varying the parameters simulta-neously. Four independent variables were X1: enzyme loading, X2:oil to methyl acetate molar ratio, X3: temperature and X4: reactiontime. The response chosen was fatty acid methyl ester (FAME)yields which were obtained from the reaction. Table 1 shows theranges and levels of the four independent variables with actual

e methodology assisted biodiesel production from waste cooking oil using1016/j.wasman.2015.07.036

Fig. 1. Biodiesel production using lipase as the biocatalyst (Adapted from Du et al., 2004).

Table 1Experimental range and levels of independent variables biodiesel production.

Code Variables �2 �1 0 +1 +2

X1 Enzyme loading (g) 1.5 1.75 2 2.25 2.5X2 Oil to methyl acetate molar ratio 1:10 1:11 1:12 1:13 1:14X3 Temperature (�C) 30 32.5 35 37.5 40X4 Reaction time (h) 48 54 60 66 72

Table 2Experimental design for interesterification of waste cooking oil (WCO).

Run X1 X2 X3 X4 Biodiesel yield (wt.%)

ExperimentalFAME (wt.%)

PredictedFAME (wt.%)

1 �1.00 �1.00 �1.00 �1.00 79.38 78.402 0.00 0.00 �2.00 0.00 84.81 81.833 0.00 0.00 0.00 0.00 93.61 93.614 1.00 1.00 1.00 1.00 68.13 68.895 1.00 �1.00 1.00 �1.00 68.98 66.606 �1.00 1.00 �1.00 1.00 80.95 83.117 1.00 �1.00 �1.00 �1.00 70.01 71.498 �1.00 1.00 �1.00 �1.00 77.54 76.379 1.00 1.00 �1.00 �1.00 62.93 67.50

10 0.00 0.00 0.00 0.00 93.61 93.6111 0.00 0.00 0.00 0.00 93.61 93.6112 �1.00 �1.00 1.00 1.00 66.66 61.8713 1.00 �1.00 �1.00 1.00 74.90 74.4714 �1.00 �1.00 �1.00 1.00 82.67 80.7115 1.00 �1.00 1.00 1.00 69.98 71.0216 0.00 0.00 0.00 �2.00 78.29 77.3817 �1.00 1.00 1.00 �1.00 53.32 53.5318 1.00 1.00 �1.00 1.00 72.95 74.9119 0.00 0.00 0.00 0.00 93.61 93.6120 0.00 �2.00 0.00 0.00 67.21 72.0921 �1.00 1.00 1.00 1.00 63.32 61.7122 1.00 1.00 1.00 �1.00 58.21 60.0423 �2.00 0.00 0.00 0.00 50.44 55.3824 2.00 0.00 0.00 0.00 60.24 55.6525 0.00 0.00 0.00 0.00 93.61 93.61

S.A. Razack, S. Duraiarasan / Waste Management xxx (2015) xxx–xxx 3

and coded values of each parameter. The independent variables arecoded to two levels namely: low (�1) and high (+1), whereas theaxial points are coded as �2 and +2. All reactions were performedin a 1000 ml screw-capped glass vessel under continuous stirring(250 rpm).

A five-level-four-factor central composite design was employedto fit a second-order response surface model which required 30experiments, including 16 factorial points, eight axial points andsix replicates at the center points, which are used to determinethe experimental error (pure error) and the reproducibility of thedata. The complete CCD design matrix in terms of real and codedindependent variable is presented and the corresponding resultsare given in Table 2. The experiments were run in replicates tominimize errors. The biodiesel yield was calculated as Eq. (1);

Biodiesel yieldð%Þ ¼ grams of biodiesel producedgrams of oil used inreaction

� 100 ð1Þ

26 �1.00 �1.00 1.00 �1.00 60.22 58.1327 0.00 0.00 2.00 0.00 52.21 55.5428 0.00 0.00 0.00 0.00 93.61 93.6129 0.00 2.00 0.00 0.00 72.47 67.9430 0.00 0.00 0.00 2.00 87.28 88.54

2.6. Statistical analysis

The experimental data obtained from CCD were analyzed byRSM. A mathematical model, following a second-order polynomialEq. (2) which includes all interaction terms was used to calculatethe predicted response:

l ¼ b0 þX4

i¼1

biXi þX4

i¼1

biiX2i þ

X3

i¼1

X4

j¼iþ1

bijXiXj þX4

i¼1

biiiX3i ð2Þ

where l is the yield of biodiesel from waste cooking oil, b0 is theoffset term, bi is the linear effect, bii is the squared effect, bij is theinteraction effect, xi is the ith independent variable and xj is thejth independent variable. The data were analyzed using DesignExpert program (version 7.0.0) and the coefficients were interpretedusing F-test. Analysis of variance (ANOVA), regression analysis andplotting of contour plot were used to establish the optimum condi-tions for the yield of waste cooking oil methyl ester. The accuracyand general ability of the above polynomial model could beevaluated by the coefficient of determination R2. The experimentwas carried out in triplicates.

Please cite this article in press as: Razack, S.A., Duraiarasan, S. Response surfacencapsulated mixed enzyme. Waste Management (2015), http://dx.doi.org/10.

3. Results and discussion

3.1. Lipase production and activity

B. cepacia (MTCC 1617) and B. subtilis was used for lipaseproduction at an optimum condition of 48 h at 37 �C and200 rpm. Different concentrations of olive oil such as 1%, 2%, 3%and 4% were tested to enhance lipase production. However, 2%only gave maximum lipase yield and such concentration was usedin production medium for lipase production. The lipase activityfrom mixed culture supernatant was found to be 17.8 U/ml.

3.2. Development of regression model

In the current research work, the relationship between responseWCO methyl esters and four reaction variables such as effect of bio-catalyst loading, oil to methyl acetate molar ratio, temperature and

e methodology assisted biodiesel production from waste cooking oil using1016/j.wasman.2015.07.036

4 S.A. Razack, S. Duraiarasan / Waste Management xxx (2015) xxx–xxx

reaction time were evaluated by using response surface methodol-ogy (RSM). The results at each point based on the experimental cen-tral composite (ECC) design and their corresponding predictedvalues are presented in Table 2. The statistical software wasemployed to determine and evaluate the coefficients of the fullregression model equation and their statistical significance.Regression analysis was employed to fit the empirical model withthe generated response variable data. The second degree polynomialmodel developed for the yield of FAME was regressed as represented

Y ¼ 93:61þ 0:068A� 1:04B� 6:57C þ 2:79D� 0:49ABþ 3:85AC

þ 0:17AD� 0:64BC þ 1:11BDþ 0:36CD� 9:52A2 � 5:9B2

� 6:23C2 � 2:66D2

where Y is the biodiesel yield, A is the enzyme load, B is molar ratio,C is temperature and D is time.

The result of statistical analysis of variance (ANOVA) which wascarried out to determine the significance and fitness of the quadra-tic model as well as the effect of significant individual terms andtheir interaction on the selected responses are presented inTable 3. The goodness of fit of regression equation developed couldbe measured by adjusted determination coefficient. The R2 value of0.9640 and adjusted R2 of 0.9304 shows that the model could besignificant predicting the response and explaining 95% of the vari-ability in the EPS synthesis. The Predicted R2 of 0.7928 is in reason-able agreement with the ‘‘Adjusted R2 of 9304. The statisticalsignificance of the equation was evaluated by F-test and ANOVA(analysis of variance) which showed that the model was statisti-cally significant at 95% confidence level (p < 0.0001). ANOVAreported the model F-value of 28.71 implying that the model is sig-nificant. p-value denotes the importance of each coefficient, help-ing in understanding the interactions among the variables. Themost significant factors of this model are X3, X1

2, X22 and X3

2. p-valuesgreater than 0.1000 indicate the model terms are not significant.The model also depicted the statistically non significant lack offit (p > 0.05), indicating that the responses are adequate foremploying in this model.

3.3. Optimization of interesterification parameters

Fig. 2 shows that surface and contour plots of biodiesel yieldfrom WCO under effect important variables such as enzyme

Table 3Analysis of variance (ANOVA) Response Surface Quadratic model.

Source Sum of squares df

Model 5111.03 14A-enzyme 0.11 1B-molar ratio 25.90 1C-temperature 1036.35 1D-time 186.76 1AB 3.85 1AC 236.62 1AD 0.45 1BC 6.59 1BD 19.65 1CD 2.07 1A2 2487.55 1B2 954.22 1C2 1064.83 1D2 194.36 1Residual 190.76 15Lack of fit 190.76 10Pure error 0.000 5Cor total 5301.79 29

Please cite this article in press as: Razack, S.A., Duraiarasan, S. Response surfacencapsulated mixed enzyme. Waste Management (2015), http://dx.doi.org/10.

loading, oil/methyl acetate molar ratio, temperature and reactiontime. The contour lines of biodiesel yield helps to understandhow the biodiesel yield changes when the experimental conditionsare changed (Muppaneni et al., 2013). There is a considerable dif-ference in the percentage yield of biodiesel from WCO under thedifferent conditions (Table 2).

The amount of enzyme used for biodiesel is a crucial factor forsuccessful industrial applications (Jiang et al., 2014). The influenceof the biocatalyst amount in the reaction yield is evaluated byincreasing the immobilised beads from 1.5 to 2.5 g. Thus, a linearincrease in the reaction conversion with increasing amount ofimmobilised bead to a maximum yield of 93.61% with 2 g(Table 2). Current finding was compared with Dizge et al. (2009)and the study resulted in only 50.9% of biodiesel from WCO usingimmobilized single lipase from T. lanuginosus. Similarly Gharat andRathod (2013) obtained 85% from the same substrate by the immo-bilised lipase from Geotrichum sp. This difference might be due todifferent specificities of two enzymes which hydrolyse the sub-strate at different regions produces higher conversion rate(Rodrigues and Ayub, 2011). But the percentage of biodiesel yieldis reduced when the lipase amount reached beyond 2 g. This wasalso reported by Li and Yan (2010), whose study was based uponthe transesterification of Sapium sebiferum oil. This may be dueto the fact that in the presence of a high amount of lipase, theactive site cannot be exposed to the substrates and many mole-cules of the enzyme aggregate together (Calero et al., 2014).

The molar ratio of oil to methyl acetate is one of the mostimportant parameters in biodiesel production. Generally transes-terification or interesterification is reversible reactions which needat least three moles of acyl acceptor to produce three moles of esterfor a reaction with one mole of oil (Zarei et al., 2014). As can beseen in Fig. 2, the yield of biodiesel increased with increasing molarratio to 1:12 and then decreased significantly. Altering the molarratio above or below the optimum value would decrease the bio-diesel yield (Jiang et al., 2014). This is due to the excess amountof methyl acetate that diluted the reaction mixture resulted inlow percentage yield of biodiesel (Xu et al., 2013).

The biodiesel yield was enhanced with the increase in reactiontemperature and then reduction trend in the yield of biodiesel canbe seen after the optimum temperature (Zarei et al., 2014). Most ofthe enzymatic transesterification depends on temperature whichenhances the activity of immobilised lipase, reaction rate and yieldof fatty acid methyl esters. Meanwhile, high temperature improved

Mean square F value Prob > F

365.07 28.71 <0.00010.11 8.705E�003 0.9269

25.90 2.04 0.17411036.35 81.49 <0.0001

186.76 14.69 0.00163.85 0.30 0.5902

236.62 18.61 0.00060.45 0.036 0.85306.59 0.52 0.4826

19.65 1.54 0.23302.07 0.16 0.6926

2487.55 195.60 <0.0001954.22 75.03 <0.0001

1064.83 83.73 <0.0001194.36 15.28 0.0014

12.7219.08

0.000

e methodology assisted biodiesel production from waste cooking oil using1016/j.wasman.2015.07.036

48 54

60 66

72

1.5 1.7

1.9 2.1

2.3 2.5

50

60

70

80

90

100

biod

iese

l yie

ld

A: enzyme (g)D: time (h)10

11

12

13

14

1.5 1.7

1.9 2.1

2.3 2.5

50

60

70

80

90

100

biod

iese

l yie

ld

A: enzyme (g)B: molar ratio

30

32

34

36

38

40

1.5

1.7

1.9

2.1

2.3

2.5

50

60

70

80

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100

biod

iese

l yie

ld

A: enzyme (g)C: temperature (C)

30

32

34

36

38

40

10

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12

13

14

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biod

iese

l yie

ld

B: molar ratioC: temperature (C)

48

54

60

66

72

10

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14

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biod

iese

l yie

ld

B: molar ratioD: time (h)

48

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72

30 32

34 36

38 40

50

60

70

80

90

100

biod

iese

l yie

ld

C: temperature (C)D: time (h)

Fig. 2. Response surface and contour plots of variables affecting biodiesel yield from WCO.

S.A. Razack, S. Duraiarasan / Waste Management xxx (2015) xxx–xxx 5

the dispersion of catalyst particle in liquid medium with bettermass transfer between the reactants (Lee et al., 2011), but the yieldis reduced when the temperature is elevated (Fig. 2). This is due tothermal denaturation of the enzyme might occur with elevation of

Please cite this article in press as: Razack, S.A., Duraiarasan, S. Response surfacencapsulated mixed enzyme. Waste Management (2015), http://dx.doi.org/10.

temperature, thus transformation of oil to FAME gets negativelyaffected (Lee et al., 2013; Rahimi et al., 2014; Jiang et al., 2014;Zarei et al., 2014; Cervero et al., 2014). Tran et al. (2012) examinedthe effect of temperature in the range of 25–40 �C on biodiesel

e methodology assisted biodiesel production from waste cooking oil using1016/j.wasman.2015.07.036

808284868890929496

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Bio

dies

el y

ield

(%)

Number of cycles

Fig. 3. Stability and reusability of immobilised mixed lipase.

6 S.A. Razack, S. Duraiarasan / Waste Management xxx (2015) xxx–xxx

from Chlorella vulgaris ESP-31using immobilised Burkholderialipase. The study resulted in highest biodiesel yield as 68.72% at40 �C, but the conversion declined when the temperature furtherincreased to 50 �C. In the present study, the temperature was notexceeded more than 40 �C, because the immobilised materialsodium alginate dissolves at higher temperature, thereby reducingthe energy consumption since higher temperature had not beenimplemented.

Reaction time is one of the important experimental factorswhich play a crucial role which is related to economic aspectsand energy consumption (Balat and Balat, 2010; Muppaneniet al., 2013). From the experiments and the statistical analysis itis clear that a minimum biodiesel yield can be achieved in a lowerreaction time but it reaches a maximum of 93.61% in 60 h (Table 2).Further increase in reaction time did not affect the biodiesel yield.This is due to the reverse reaction leading to loss of fatty acidmethyl ester formation (Jeong and Park, 2008; Leung et al.,2010). So a reaction time of 60 h was taken as the optimumreaction time for maximum biodiesel yield.

The reusability is one of the important factors to heterogeneouscatalysts for industry application (Yan et al., 2014; Jiang et al.,2014; Arumugam and Ponnusami, 2014). This factor decides thepossibility of large scale production of biodiesel utilizing enzymes(Gharat and Rathod, 2013). After each batch run, immobilisedbeads were separated by simple filtration, and then washed withmethyl acetate, dried and reused. Fig. 3 showed that there is no sig-nificant loss of lipase activity even after 20 reuse of immobilisedmixed enzyme. Generally biocatalyst is inactivated by methanolafter four or five usage of lipase and which is one of major limita-tions in lipase mediated biodiesel production. But in the currentstudy acyl acceptor methyl acceptor did not affect the lipase, hencelipase can be used number of cycles when applying methyl acetateinstead of methanol or ethanol. This is in agreement with Du et al.(2004) showed that there was no loss of biocatalyst even after onehundred cycles of repeated usage in the presence of methylacetate.

3.4. Validation of the model

The optimized conditions to produce waste cooking oil methylesters were obtained from the regression model. In order to con-firm the accuracy of the model for lipase catalyzed interesterifica-tion of the waste cooking oil, experiments were carried out underoptimized conditions. The following optimal reaction conditions,biocatalyst loading (2 g), oil/methyl acetate molar ratio (1:12) tem-perature (35 �C), time (60 h) were determined using the regressionmodel. Triplicate experiments were carried out to verify and vali-date the regression model. The average yield of biodiesel obtainedduring the experiment was found to be 93.61%. The predicted valuewas calculated to be 93.78% through the statistical technique, with

Please cite this article in press as: Razack, S.A., Duraiarasan, S. Response surfacencapsulated mixed enzyme. Waste Management (2015), http://dx.doi.org/10.

a minimal error of 0.18%. As the experimental and predicted valueswere in close agreement with each other, the model was consid-ered to be highly appropriate and accurate for producing FAMEfrom waste cooking oil.

4. Conclusion

This work was carried out to investigate interesterification ofwaste cooking oil to methyl esters using immobilised lipase. Theoptimization of this experiment was performed statisticallythrough Response Surface Methodology. Lipase from mixed cul-tures of B. cepacia and B. subtilis were used, in immobilised form,for upgrading the production of FAME. The optimized values,obtained from Central Composite Design, of the parametersnamely enzyme load, oil/methyl acetate molar ratio, temperatureand reaction time for the process were 2 g, 1:12, 35 �C and 60 hrespectively. Immobilization of the enzyme had revealed its stabil-ity even after 20 cycles of repeated usage, demonstrating thatimmobilization is an advantageous technique to mitigate the costof the process. The study indicated that the optimization protocolis an essential way to improve and enhance the process of convert-ing WCO to fatty acid methyl esters for better production of envi-ronmental oriented biodiesel in a large scale operation.

Acknowledgment

The authors cordially thanks to Mr. Saranraj, Ph.D. ResearchScholar, Department of Microbiology, Faculty of Agriculture,Annamalai University, Tamilnadu, India for providing bacterial cul-tures to carry out this research work.

References

Adachi, D., Hama, S., Nakashima, K., Bogaki, T., Ogino, C., Kondo, A., 2013. Productionof biodiesel from plant oil hydrolysates using an Aspergillus orzyae whole-cellbiocatalyst highly expressing Candida antarctica lipase B. Bioresour. Technol.135, 410–416.

Antczak, M.S., Kubiak, A., Antczak, T., Bielecki, S., 2009. Enzymatic biodieselsynthesis – key factors affecting efficiency of the process. Renew. Energy 34,1185–1194.

Arumugam, M., Ponnusami, V., 2014. Biodiesel production from Calophylluminophyllum oil using lipase producing Rhizopus oryzae cells immobilizedwithin reticulated foams. Renew. Energy 64, 276–282.

Balat, M., Balat, H., 2010. Progress in biodiesel processing. Appl. Energy 87, 1815–1835.

Burkert, J.F.M., Maugeri, F., Rodrigues, M.I., 2004. Optimization of extracellularlipase production by Geotrichum sp. using factorial design. Bioresour. Technol.91, 77–84.

Calero, J., Verdugo, C., Luna, D., Sancho, E.D., Luna, C., Posadillo, A., Bautista, F.M.,Romero, A.A., 2014. Selective ethanolysis of sunflower oil with Lipozyme RM IMan immobilised Rhizomucor miehei lipase, to obtain a biodiesel like biofuel,which avoids glycerol production through the monoglyceride formation. NewBiotechnol. 31, 596–601.

Campanelli, P., Banchero, M., Manna, L., 2010. Synthesis of biodiesel from edible,non-edible and waste cooking oils via supercritical methyl acetatetransesterification. Fuel 89, 3675–3682.

Casas, A., Ramos, M.J., Pérez, A., 2011. Kinetics of chemical interesterification ofsunflower oil with methyl acetate for biodiesel and triacetin production. Chem.Eng. J. 171, 1324–1332.

Cervero, J.M., Alvarez, J.R., Luque, S., 2014. Novozym 435-catalyzed synthesis offatty acid ethyl esters from soybean oil for biodiesel production. BiomassBioenergy 61, 131–137.

Chhetri, A.B., Watts, K.C., Islam, M.R., 2008. Waste cooking oil as an alternatefeedstock for biodiesel production. Energies 1, 3–18.

Dalal, S., Singh, P.K., Raghava, S., Rawat, S., Gupta, M.N., 2008. Purification andproperties of the alkaline lipase from Burkholderia cepacia ATCC 25609.Biotechnol. Appl. Biochem. 51, 23–31.

Dizge, N., Aydiner, C., Imer, D.Y., Bayramoglu, M., Tanriseven, A., Keskinler, B., 2009.Biodiesel production from sunflower, soybean, and waste cooking oils bytransesterification using lipase immobilized onto a novel microporous polymer.Bioresour. Technol. 100, 1983–1991.

Du, W., Xu, Y., Liu, D., Zeng, J., 2004. Comparative study on lipase-catalysedtransesterification of soybean oil for biodiesel production withdifferent acyl acceptors. J. Mol. Catal. B Enzym. 3, 125–129.

e methodology assisted biodiesel production from waste cooking oil using1016/j.wasman.2015.07.036

S.A. Razack, S. Duraiarasan / Waste Management xxx (2015) xxx–xxx 7

Garcia, R., Calvo, L., Ladero, M., 2011. Enzymatic synthesis of biodiesel frommicroalgal model oil: optimization of operational conditions. In: 9th GreenChemistry Conference, An international Event, Alcala de Henares-Spain.

Gharat, N., Rathod, V.K., 2013. Ultrasound assisted enzyme catalyzedtransesterification of waste cooking oil with dimethyl carbonate. Ultrason.Sonochem. 20, 900–905.

Gnanaprakasam, A., Sivakumar, V.M., Surendhar, A., Thirumarimurugan, M.,Kannadasan, T., 2013. Recent strategy of biodiesel production from wastecooking oil and process influencing parameters: a review. http://dx.doi.org/10.1155/2013/926392.

Gumbyte, M., Makareviciene, V., Sendzikiene, E., 2011. Esterification of by-productsof biodiesel fuel production with methanol and technical glycerol usingbiocatalysts. Environ. Eng. Manage 2 (56), 28–34.

Jeong, G.T., Park, D.H., 2008. Lipase-catalyzed transesterification of rapeseed oil forbiodiesel production with tert-butanol. Appl. Biochem. Biotechnol. 1 (S148),131–139.

Jiang, Y., Liu, X., Chen, Y., Zhou, L., He, Y., Ma, L., Gao, J., 2014. Pickering emulsionstabilized by lipase-containing periodic mesoporous organosilica particles: arobust biocatalyst system for biodiesel production. Bioresour. Technol. 153,278–283.

Kawakami, K., Oda, Y., Takahashi, R., 2011. Application of a Burkholderia cepacialipase immobilized silica monolith to batch and continuous biodieselproduction with a stoichiometric mixture of methanol and crude Jatropha oil.Biotechnol. Biofuels 4 (42), 1–11.

Kuan, I.C., Lee, C.C., Tsai, B.H., Lee, S.L., Lee, W.T., Yu, C.Y., 2013. Optimizing theproduction of biodiesel using lipase entrapped in biomimetic silica. Energies 6(4), 2052–2064.

Lee, H.V., Yunus, R., Juan, J.C., Taufiq-Yap, Y.H., 2011. Process optimization design forjatropha-based biodiesel production using response surface methodology. FuelProcess. Technol. 92, 2420–2428.

Lee, O.K., Kim, Y.H., Na, J.G., Oh, Y.K., Lee, E.Y., 2013. Highly efficient extraction andlipase-catalyzed transesterification of triglycerides from Chlorella sp. KR-1 forproduction of biodiesel. Bioresour. Technol. 147, 240–245.

Leung, D.Y.C., Wu, X., Leung, M.K.H., 2010. A review on biodiesel production usingcatalyzed transesterification. Appl. Energy 87, 1083–1095.

Li, Q., Yan, Y., 2010. Production of biodiesel catalysed by immobilized Pseudomonascepacia lipase from Sapium sebiferum oil in micro-aqueous phase. Appl. Energy87, 3148–3154.

Maddikeri, G.L., Pandit, A.B., Gogate, P.R., 2013. Ultrasound assistedinteresterification of waste cooking oil and methyl acetate for biodiesel andtriacetin production. Fuel Process. Technol. 116, 241–249.

Please cite this article in press as: Razack, S.A., Duraiarasan, S. Response surfacencapsulated mixed enzyme. Waste Management (2015), http://dx.doi.org/10.

Modi, M.K., Reddy, J.R.C., Rao, B.V.S.K., Prasad, R.B.N., 2007. Lipase-mediatedconversion of vegetable oils into biodiesel using ethyl acetate as acylacceptor. Bioresour. Technol. 98, 1260–1264.

Muppaneni, T., Reddy, H.K., Ponnusamy, S., Patil, P.D., Sun, Y., Peter Dailey, P., Deng,S., 2013. Optimization of biodiesel production from palm oil under supercriticalethanol conditions using hexane as co-solvent: a response surface methodologyapproach. Fuel 107, 633–640.

Omar, W.N.N.W., Amin, N.A.S., 2011. Biodiesel production from waste cooking oilover alkaline modified zirconia catalyst. Fuel Process. Technol. 92, 2397–2405.

Padilha, G.S., Curvelo Santana, J.S., Alegre, R.M., Tambourgi, E.B., 2012. Extraction oflipase from Burkholderia cepacia by PEG/phosphate ATPS and its biochemicalcharacterization. Braz. Arch. Biol. Technol. 55, 7–19.

Rahimi, M., Aghel, B., Alitabar, M., Sepahvand, A., Ghasempour, H.R., 2014.Optimization of biodiesel production from soybean oil in a microreactor.Energy Convers. Manage. 79, 599–605.

Rodrigues, R.C., Ayub, M.A. Zachia, 2011. Effects of the combined use ofThermomyces lanuginosus and Rhizomucor miehei lipases for thetransesterification and hydrolysis of soybean oil. Process Biochem. 46, 682–688.

Sathasivam, S., Manickam, A., 1996. Biochemical Methods, second ed. New AgeInternational Pvt Ltd., New Delhi, India, pp. 23–25.

Sivaramakrishnan, R., Muthukumar, K., 2012. Isolation of thermostable and solvent-tolerant Bacillus sp. lipase for the production of biodiesel. Appl. Biochem.Biotechnol. 166, 1095–1111.

Tran, D.T., Yeh, K.L., Chen, C.L., Chang, J.S., 2012. Enzymatic transesterification ofmicroalgal oil from Chlorella vulgaris ESP-31 for biodiesel synthesis usingimmobilized Burkholderia lipase as the biocatalyst. Bioresour. Technol. 108,119–127.

Xu, H., Miao, X., Wu, Q., 2006. High quality biodiesel production from a microalgaChlorella protothecoides by heterotrophic growth in fermenters. J. Biotechnol.126, 499–507.

Xu, Y., Du, W., Liu, D., Zeng, J., 2013. A novel enzymatic route for biodieselproduction from renewable oils in a solvent-free medium. Biotechnol. Lett. 25,1239–1241.

Yan, J., Zheng, X., Li, S., 2014. A novel and robust recombinant Pichia pastoris yeastwhole cell biocatalyst with intracellular over expression of a Thermomyceslanuginosus lipase: Preparation, characterization and application in biodieselproduction. Bioresour. Technol. 151, 43–48.

Zarei, A., Amin, N.A.S., Kiakalaieh, A.T., Zain, N.A.M., 2014. Immobilized lipase-catalyzed transesterification of Jatropha curcas oil: optimization and modeling.J. Taiwan Inst. Chem. Eng. 45, 444–451.

e methodology assisted biodiesel production from waste cooking oil using1016/j.wasman.2015.07.036