Bioresource Technology, Volume 100, Issue 6, March 2009, Pages 1983-1991

Biodiesel production from sun?ower,soybean,and waste cooking oils by

transesteri?cation using lipase immobilized onto a novel microporous polymer

Nadir Dizge a ,Coskun Aydiner a ,Derya Y.Imer a ,Mahmut Bayramoglu b ,Aziz Tanriseven c ,Bülent Keskinler a,*

a

Gebze Institute of Technology,Department of Environmental Engineering,Gebze 41400,Turkey b

Gebze Institute of Technology,Department of Chemical Engineering,Gebze 41400,Turkey c

Gebze Institute of Technology,Department of Biochemistry,Gebze 41400,Turkey

a r t i c l e i n f o Article history:

Received 22July 2008

Received in revised form 10October 2008Accepted 12October 2008

Available online 22November 2008Keywords:Biodiesel

Transesteri?cation

Microporous polymeric biocatalyst Thermomyces lanuginosus lipase Taguchi methodology

a b s t r a c t

This study aims at carrying out lipase-catalyzed synthesis of fatty acid methyl esters (biodiesel)from various vegetable oils using lipase immobilized onto a novel microporous polymeric matrix (MPPM)as a low-cost biocatalyst.The research is focused on three aspects of the process:(a)MPPM synthesis (monolithic,bead,and powder forms),(b)microporous polymeric biocatalyst (MPPB)preparation by immobilization of lipase onto MPPM,and (c)biodiesel production by MPPB.Experimental planning of each step of the study was separately carried out in accordance with design of experiment (DoE)based on Taguchi methodology.

Microporous polymeric matrix (MPPM)containing aldehyde functional group was synthesized by polyHIPE technique using styrene,divinylbenzene,and polyglutaraldehyde.Thermomyces lanuginosus lipase was covalently attached onto MPPM with 80%,85%,and 89%immobilization ef?ciencies using bead,powder,and monolithic forms,respectively.Immobilized enzymes were successfully used for the production of biodiesel using sun?ower,soybean,and waste cooking oils.It was shown that immo-bilized enzymes retain their activities during 10repeated batch reactions at 25°C,each lasting 24h.Since the developed novel method is simple yet effective,it could have a potential to be used industrially for the production of chemicals requiring immobilized lipases.

ó2008Elsevier Ltd.All rights reserved.

1.Introduction

In recent years,demand for fatty acid methyl esters (biodiesel)being used as fuel in diesel engines and heating systems increased due to recent rises in petroleum prices,increments in earth popula-tion and energy necessity,and development of government mea-sures such as The EU Directive 2003/30/EC and The US Energy Policy Act (EPAct,1992)(Vicente et al.,2007).Transesteri?cation of vegetable oils to obtain biodiesel consists in replacing the glycerol of triglycerides with a short chain alcohol in the presence of a cata-lyst.Transesteri?cation reaction can be catalyzed by both homoge-neous (basic or acidic)and heterogeneous (basic,acidic or enzymatic)catalysts (Mittelbach,1990).Recent studies show that biodiesel can be produced enzymatically by lipase-catalyzed transe-steri?cation which has become more attractive in biodiesel produc-tion since the glycerol can be recovered easily and the puri?cation process for biodiesel is simple (Vicente et al.,1998).In addition,the use of lipase in biodiesel production tolerates the water content of oil and increases biodiesel yield by avoiding the soap formation.

Selection of an immobilization strategy greatly in?uences the properties of biocatalyst.The decrement levels in activity and dif-fusion limitations occurring with immobilization are mainly dependent on the properties of support material and the immobi-lization method.Support materials playing an important role in the usefulness of an immobilized enzyme should be low-cost,and pro-vide adequate large surface area together with the least diffusion limitation in the transport of substrate and product for enzymatic reactions (Arica et al.,2000).Enzymatic biodiesel production was studied using lipases immobilized onto different supports such as macroporous resin by adsorption (Yang et al.,2006),hydrophobic sol–gel support by entrapment (Noureddini et al.,2005),silica aerogel by encapsulation (Or?aire et al.,2006)and chitin by chem-ical binding (Gomes et al.,2004).

The present work is on immobilization of Thermomyces lanugi-nosus lipase onto a novel microporous polymer in different forms (bead,powder,and monolithic)having functional aldehyde groups and its usage for biodiesel synthesis using sun?ower,soybean,and waste cooking oils.The novel polymer synthesis,lipase immobili-zation,and biodiesel production were planned using Taguchi 2n designs since a great number of independent process variables are involved.

0960-8524/$-see front matter ó2008Elsevier Ltd.All rights reserved.doi:10.1016/j.biortech.2008.10.008

*Corresponding author.Tel.:+902626053207;fax:+902626053205.E-mail address:bkeskinler@https://www.wendangku.net/doc/5b8453491.html,.tr (B.Keskinler).Bioresource Technology 100(2009)

1983–1991

Contents lists available at ScienceDirect

Bioresource Technology

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /b i o r t e c

h

2.Methods

2.1.Materials

2.1.1.Chemicals

Styrene(STY),divinylbenzene(DVB),and glutaraldehyde(GA) (25%w/v)were obtained from Merck.Sorbitan monooleate(Span 80)and p-nitrophenyl palmitate(p-NPP)were from Fluka.Potas-sium persulphate was purchased from Riedel-de-Haen.Vegetable oils(sun?ower,soybean,and waste cooking oil)were obtained locally.The composition of the oils with regard to fatty acid con-tent was determined by gas chromatography(GC)in accordance with The American Oil Chemical Society of?cial methods(AOCS, 1998)and the results were presented in Table1.

2.1.2.Enzyme

Commercial soluble lipase from https://www.wendangku.net/doc/5b8453491.html,nuginosus was purchased from Novo Nordisk(Denmark).It is produced by submerged fer-mentation of a genetically modi?ed Aspergillus oryzae microorgan-ism.The enzyme activity was16.3U/ml.One unit of activity(U)is de?ned as the amount of enzyme forming1l mol p-nitrophenol per minute from p-nitrophenyl palmitate using lipase assay method.Protein determinations were carried out using Bradford method(Bradford,1976).

2.2.Experimental procedure

All statistical analysis in polymer synthesis was performed for only monolithic forms.Chosen factor levels and values of all vari-ables in Taguchi design are shown in Table2.Lipases immobilized onto matrixes(bead,powder or monolithic form)were used for biodiesel synthesis and the results obtained were compared.

2.2.1.Polyglutaraldehyde(PGA)synthesis

PGA was synthesized by polymerizing glutaraldehyde solution (25%w/v),pH of which was10.5by adding NaOH solution(1M), at room temperature for30min and neutralizing the mixture using HCl solution(1M).

2.2.2.Preparation of MPPM in monolithic form and immobilization of lipase using Taguchi methodology

MPPM was prepared by polymerizing the continuous phase of a high internal phase emulsion consisting of organic and water phases.The compositions of these phases are shown in Table2.

The organic phase(5ml)was composed of styrene,divinylben-zene,and Span80.The water phase(45ml)contained potassium persulphate and polyglutaraldehyde solution.Span80(1.75g) and potassium persulphate(0.7g)were used in all samples pre-pared.The aqueous phase was added dropwise to the organic phase while stirring with a mechanical stirrer based on the condi-tions in Table2.The emulsion was transferred to a mold and cured at80°C for24h(Dizge et al.,2008).The polymer was?rst washed with distilled water(50ml)and then with tert-butanol(50ml)to remove impurities.

Immobilization was carried out by circulating enzyme solution in calcium acetate buffer(55ml,25mM,pH6)by a peristaltic pump through microporous monolithic polymer in disk form (m=0.36g,D=2cm,and h=0.5cm)in a holder made of stainless steel at various temperatures and immobilization time(Fig.1a). The immobilized enzyme was then washed with acetate buffer (50ml,25mM,pH6)to remove the unbound enzyme.The super-natant and washing solutions were assayed for unbound protein. SEM micrographs of microporous polymeric material(MPPM) and microporous polymeric biocatalyst(MPPB)for monolithic form are shown in Fig.1c.

2.2.

3.Preparation of MPPM in powder and bead forms and their usage in lipase immobilization

Powder form in a range of4–8mesh in size was prepared from the monolithic polymer produced using medium values(Table2). Beads were synthesized by modi?cation of the work of Desforges et al.,in which the initiator,potassium persulphate,was used in polyglutaraldehyde solution(Desforges et al.,2002).

Immobilization of lipase was carried out by reaction of powder or bead matrix(1g)with enzyme(1ml)in calcium acetate buffer

Table1

Fatty acid compositions of the sun?ower,soybean,and waste cooking oil.

Fatty acid oil Sun?ower oil Soybean oil Waste cooking oil

Lauric(12:0)––0.05

Myristic(14:0)0.060.070.19

Palmitic(16:0) 5.6810.878.90

Palmitoleic(16:1)0.140.100.22

Stearic(18:0) 3.61 3.66 3.85

Oleic(18:1)34.2723.5930.71

Linoleic(18:2)54.7953.8654.35

Linolenic(18:3)0.07 6.490.27

Arachidic(20:0)0.250.370.29

Gadoleic(20:1)0.130.220.18

Behenic(22:0)0.690.450.61

Lignoceric(24:0)0.230.180.24

Table2

Factor levels and values of all variables in Taguchi designs applied during each of three consecutive experimentations.

Process Factors Parameter Levels and values

Low(à1)Medium(0)High(+1)

MPPM synthesis X1STY/DVB ratio(vol./vol.%)a0 2.55

X2PGA concentration(vol.%)b1815

X3Monomers dozing time(min)51321

X4Mixing time(min)204060

X5Mixing rate(rpm)300450600

MPPB synthesis X6Lipase concentration(vol.%)1 5.510

X7Immobilization temperature(°C)41526

X8Immobilization time(h)22548

X9Enzyme?ow(ml/min)11121

Biodiesel production X10Substrate?ow(ml/min)11121

X11Reaction temperature(°C)355065

X12Alcohol:oil ratio(mol/mol)4:15:16:1

X13Water amount(%)1815

X14Total reaction time(h)135

a Organic phase(5ml)contains Span80(0.75ml)and a mixture of STY and DVB(4.25ml).

b Water phase(45ml)contains PGA(0.45,3.6,and6.75ml)and potassium persulphate(0.7g).

1984N.Dizge et al./Bioresource Technology100(2009)1983–1991

(19ml,25mM,pH 6)at 26°C for 25h with gentle shaking (250rpm).The immobilized enzyme was then washed with ace-tate buffer (50ml)to remove the unbound enzyme.The superna-tant and washing solutions were assayed for unbound protein.2.2.4.Biodiesel production by microporous polymeric biocatalyst (MPPB)in monolithic form using Taguchi methodology

Biodiesel production was studied using MPPB in monolithic form which was prepared using medium values (Table 2).The pro-duction was carried out using the system shown in Fig.1b in which the biocatalyst,MPPB,was placed in a holder.The vessel was ini-tially ?lled with the vegetable oil (92g),then placed on the con-stant-temperature mechanic stirrer (250rpm)with its associated equipment and heated to a predetermined temperature.Methanol was added to the mixture in three-steps to avoid strong methanol inhibition.Then,the mixture was recirculated through the holder for duration indicated in Table 2.Glycerol was separated from bio-diesel in a funnel within 2h.The biodiesel phase was washed with hot water twice (50ml,60°C)to remove methanol and glycerol residues.

2.2.5.Biodiesel production by MPPB in powder and bead matrix

Immobilized lipase (1g,powder or bead form)was reacted with a mixture of oil (92g)and methanol (20g)in a complete-mixing batch reactor (250rpm)at 65°C for 24h.Methanol was added to the mixture in three-steps to avoid strong methanol inhibition.After the reaction,MPPB was removed from biodiesel by ?ltration and washed with tert -butanol (30ml).

2.2.6.Determination of lipase activity

Lipase assay was performed using transesteri?cation reaction of p-nitrophenyl palmitate (p-NPP)and butanol,resulting in p-nitro-phenol (p-NP),the amount of which was determined by a colori-metric method (Soares et al.,2003).The stock solution of p-NPP

(36mM)was prepared in hexane.The reaction mixture comprised of p-NPP solution (10ml),water saturated hexane (9ml)and buta-nol (1ml).The reaction mixture was recirculated for 10min at 25°C using a peristaltic pump with a rate of 21ml/min.Reaction mixture (50l l)was added to 3ml of triethylamin-ethanol solution (3l l triethylamine per ml ethanol).p-NP formed by the enzymatic transesteri?cation reaction was determined at 410nm.Each of the assays was performed in duplicate and mean values were presented.

2.3.Analytical procedures

Tensile properties of the MPPM were determined using testing machine ‘‘Instron/5569”.The stress–strain curves of the MPPM were obtained following the standard ISO 2062(1993).

The biodiesel content in the reaction mixture was analyzed using a gas chromatograph (Agilent Technologies 6890N model)equipped with a DB-WAX (length 30m,inner diameter 0.53mm,?lm thickness 0.5l m)capillary column and a ?ame-ionization detector (FID)connected to an integraph (DIN EN 14103,2003).Mono-,di-,tri-glycerides,free and total glycerol determinations were carried out according to European standard test method of DIN EN 14105(2003).All of the analytical assays were performed in duplicate and mean values were presented.2.4.Experimental planning

This study was planned in a frame comprising three consecutive stages:(a)MPPM synthesis having mechanical stability,(b)micro-porous polymeric biocatalyst (MPPB)preparation by immobiliza-tion of lipase onto MPPM,and (c)biodiesel production by MPPB.Experiments were carried out in accordance with design of exper-iments (DoE)for more sophisticated analyses of variables with a smaller number of experiments.In experimentations,three

exper-

Fig.1.Experimental set up ((a)for the immobilization of lipase and (b)for the enzymatic production of biodiesel (c)SEM micrographs for microporous polymeric matrix (MPPM)and microporous polymeric biocatalyst (MPPB)).

N.Dizge et al./Bioresource Technology 100(2009)1983–19911985

imental structures were separately applied and all experiments were carried out using sun?ower oil.Experimental plans were se-lected according to Taguchi2n designs because of their simplicity and easiness in application.25and29partial fractional–factorial designs were used for MPPM synthesis and biodiesel production by MPPB,and MPPB synthesis,respectively.Standard Taguchi L16 and L8orthogonal arrays requiring16and eight experiments (Tables3and4)were employed in experimentations of MPPM and MPPB synthesis,and biodiesel production by MPPB,respec-tively(Ross,1996).In MPPM experiments,?ve variables(STY/ DVB ratio,PGA concentration,monomers dozing time,mixing time,and mixing rate)were taken into account for the evaluation of mechanical strength.The in?uences of nine variables were stud-ied in the synthesis of MPPB.The in?uences of lipase concentra-tion,immobilization temperature,immobilization time,and enzyme?ow on the effectiveness of MPPB synthesis were explored together with?ve variables taking place in previous stage.In addi-tion,the in?uences of alcohol to oil ratio,reaction temperature, substrate?ow,water amount,and total reaction time were also investigated for biodiesel yields.Experimental error was deter-mined by conducting three replicate experiments for the detailed statistical analyses of the results.The reliability of each experimen-tal plan was also con?rmed by the validation tests in order to determine sensibilities of the related model equations.Factor lev-els and values of all variables applied for each of experimentations were presented in Table2.

2.5.Statistical analysis

The results obtained from three consecutive stages of the study were analyzed by analysis of variance(ANOVA)using MINITAB(PA, USA)statistical software,which makes possible to interpret both singly and interactively the in?uences of input variables on the process performance.Furthermore,the percentage contribution of each input variable on each response can be comparatively determined in addition to the?rst order empirical relationships between the variables and the responses(Chen et al.,2004;Cvijo-vic et al.,2005;Ross,1996)

y?b0t

X i?p

i?1

b iáx it

X i?p

i

b ijáx i x je1T

where y is the response parameter;i and p are the variable number and the latest variable number;x i and x j are the input variables;b0, b i,and b ij are intercept,linear,and interaction parameters,respec-tively.The values of i are1,6,and10,while those of p are5,9, and14for MPPM synthesis,MPPB synthesis and biodiesel produc-tion,respectively.

3.Results and discussion

This study was planned in a frame comprising three consecutive stages:(a)MPPM synthesis(monolithic,bead,and powder forms), (b)microporous polymeric biocatalyst(MPPB)preparation by immobilization of lipase onto MPPM,and(c)biodiesel production by MPPB.Lipase from https://www.wendangku.net/doc/5b8453491.html,nuginosus was immobilized successfully onto MPPM(monolithic,bead,and powder forms)with high immobilization ef?ciencies,de?ned as the ratio of activity of immobilized enzyme to the activity of soluble enzyme used in immobilization.The immobilization ef?ciencies obtained using bead and powder forms were80%and85%,respectively.

The main aim of this study was to produce MPPB having high mechanical strength and maximum enzyme activity.The mechan-ical strength of MPPB changed in the range0.61–9.27MPa(Table 3).The immobilization ef?ciencies for MPPBs were between2%

Table3

Experimental plan by L16Taguchi design applied for MPPM and MPPB synthesis and the results.

Run MPPM synthesis MPPB synthesis

Factors Results Factors Results Results

X1X2X3X4X5Mechanical strength(MPa)X6X7X8X9Immobilization ef?ciency(%)Enzyme activity(U/g disc)

1à1à1à1à1à1 3.98à1à1+1+130.32 4.88

2à1à1à1+1à19.27+1+1à1+116.81 5.17

3à1à1+1à1+1 5.65à1+1à1+116.65 4.34

4à1à1+1+1+1 5.81+1à1+1+1 1.96 5.14

5à1+1à1à1+1 3.34+1+1à1à127.29 2.89

6à1+1à1+1+1 5.56à1à1+1à1 1.89 3.25

7à1+1+1à1à1 4.89+1à1+1à1 6.65 2.89

8à1+1+1+1à10.61à1+1à1à1 2.130.09

9+1à1à1à1+1 4.35+1à1à1à1 3.37 1.97

10+1à1à1+1+17.87à1+1+1à156.09 5.12

11+1à1+1à1à1 2.87+1+1+1à153.14 5.26

12+1à1+1+1à1 5.32à1à1à1à131.74 6.29

13+1+1à1à1à1 5.67à1+1+1+186.88 3.59

14+1+1à1+1à1 3.93+1à1à1+118.40 2.98

15+1+1+1à1+1 4.07à1à1à1+115.08 2.07

16+1+1+1+1+1 2.57+1+1+1+188.84 2.58

1700000 3.13000020.8312.83

1800000 3.27000021.1112.86

1900000 3.25000021.0712.87

Table4

Experimental plan by L8Taguchi design applied for biodiesel production by MPPB and

the results.

Run Biodiesel production

Factors Results

X10X11X12X13X14Biodiesel yield(%)

1à1à1à1+1+137.3

2à1à1+1+1à143.1

3à1+1à1à1à136.7

4à1+1+1à1+157.0

5+1+1à1+1à151.5

6+1à1+1à1à150.1

7+1à1à1à1+144.6

8+1+1+1+1+163.8

90000040.0

100000039.7

110000039.5

1986N.Dizge et al./Bioresource Technology100(2009)1983–1991

and89%.Higher mechanical strength is crucial with respect to life-time of MPPB and enzyme leakages.However,highest immobiliza-tion ef?ciency and activity per g disk were not obtained using the monolith with highest mechanical strength(9.27MPa)(Table3). As known,enzyme activity is a major parameter for reaction rate. The disk having highest activity(12.87U/g disk)had a mechanical strength of3.27MPa and this value is an acceptable one.No crush-ing was observed when the MPPB with3.27MPa was used in re-peated batch reactions.Thus,we used MPPM with moderate mechanical strength(3.27MPa)with highest activity per gram disk(12.87U/g disk)in biodiesel production.The MPPM used was prepared under medium conditions(Table2).The immobiliza-tion ef?ciency of the disk used in the study was21%,indicating that all of the enzyme is not used under the conditions studied. MPPB was used for biodiesel production and the parameters(sub-strate?ow,reaction temperature,alcohol/oil ratio,water amount, and total reaction time)were evaluated for maximum biodiesel yield.

3.1.Synthesis of MPPM

The in?uences of single and interactive variables on mechanical strength of MPPM were investigated and the results of DoEs calcu-lations are presented in Table3.Main effect plots of single vari-ables and relative in?uences of all variables are shown in Fig.2.

Determinative factors for mechanical strength of MPPM were established as single and two-way interactive variables.Various single variables(PGA concentration(X2),dozing time(X3),and mixing time(X4))noticeably in?uenced the mechanical strength of MPPM(Fig.2a).An increase in mixing time increased the mechanical strength.However,PGA concentration and dozing time had a negative effect on mechanical strength.The two-way inter-actions affected the process highly.The most in?uential ones were PGA concentration–mixing time(X2X4)and dozing time–mixing time(X3X4).The strength was also affected by three-way interac-tions among STY/DVB ratio,dozing time,and mixing time variables (X1X3X4).It is well known that during the preparation of high internal phase emulsions for the production of polyHIPE(PHP), the viscosity of emulsion increases as the mixing time and rate in-creases.It is seen from previous studies that droplet–droplet inter-actions may be due to reduced size of droplets through the increasing mixing time(Bhumgara,1995).These results showed that a polymeric matrix having more mechanical strength could be obtained by increasing the mixing time and mixing rate which led to smaller porous matrix structure.The increasing PGA concen-tration may hinder cross-linking of DVB monomers with STY ones which resulted in formation of a less strong structure.

3.2.Lipase immobilization onto MPPM

Lipase immobilization onto MPPM was carried out successfully. MPPM contains polyglutaraldehyde functional groups which react with amino,thiol,imidazole,and phenol groups of proteins(Bolgar and Gaskell,1996;Kaga et al.,1994;Weber et al.,1995).Taguchi methodology was used in?nding of immobilization conditions. Main effect plots of single variables and relative in?uences of all variables are depicted in Fig.3.

Immobilization ef?ciency was affected by STY/DVB ratio(X1), immobilization temperature(X7),immobilization time(X8),and enzyme?ow(X9).Immobilization time(X8)was the most impor-

N.Dizge et al./Bioresource Technology100(2009)1983–19911987

tant factor.However,enzyme activity was affected negatively by high PGA concentrations(X2)(Fig.3).The relative in?uences were observed in decreasing order as X1>X7>X8>X9for ef?ciency. The immobilization ef?ciency increases as STY/DVB ratio increases. It is possibly due to the formation of large pores formation.Two-way interactions affected the immobilization ef?ciency more than single variables.The most in?uential two-way interactions were PGA concentration–immobilization time(X2X8)and STY/DVB ra-tio–lipase concentration(X1–X6).

Activity of MPPB was affected by single variables(immobiliza-tion time(X8)and enzyme?ow(X9))and two-way interactive variables(STY/DVB ratio–mixing rate(X1X5),STY/DVB ratio–lipase concentration(X1X6),and PGA concentration–immobilization time(X2X8))(Fig.3).The most important variable was the immo-bilization time.

3.3.Biodiesel production by MPPB

The in?uences of single and interactive variables on biodiesel production by MPPB were studied.The results of DoEs are pre-sented in Table4.Main effect plots of single variables and relative in?uences of all variables are shown in Fig.4.

Biodiesel yield was increased signi?cantly by such single vari-ables as alcohol:oil ratio(X12),substrate?ow(X10),reaction tem-perature(X11),and total reaction time(X14)(Fig.4).However, increased amount of water(X13)had insigni?cant effect on the biodiesel yield.The important two-way interactions affecting bio-diesel yield were substrate?ow–alcohol:oil(X10X12)and sub-strate?ow–total reaction time(X10X14).Different methanol to oil ratios(4:1–6:1)were studied.It was found that an increase in alcohol to oil ratio had a positive effect on biodiesel yield.The 6:1ratio gave rise to the best result,in agreement with previous researches(Krisnangkura and Simamaharnoop,1992).

The in?uence of reaction temperature(X11)on biodiesel yield was very signi?cant in the range studied(from35to65°C).There was no decrease in activity at65°C due to thermophilic character-istic of https://www.wendangku.net/doc/5b8453491.html,nuginosus.

Previous study showed that biodiesel conversion depends on the rate of substrate?ow(Nie et al.,2006).A similar result was ob-tained in our study.Flow rate of substrate and reaction time had positive effects on biodiesel yield.This shows that the reaction was less diffusion-controlled.

It is well known that water content is an important factor affecting the activity of an enzyme in a non-aqueous medium.

1988N.Dizge et al./Bioresource Technology100(2009)1983–1991

N.Dizge et al./Bioresource Technology100(2009)1983–19911989

Table5

Regression models estimated for MPPM and MPPB synthesis and biodiesel production.

Terms Coef?cients

MPPM synthesis(mechanical strength)MPPB synthesis Biodiesel production(methyl esters yield)

Ef?ciency Activity

constant 4.75028.600 3.66047.700

X1à0.16415.6000.076?

X2à0.894 2.320à1.110?

X3à0.772à1.550à0.074?

X40.393à1.3500.171?

X50.178à2.180à0.237?

X6?à1.520à0.047?

X7?14.900à0.027?

X8?12.1000.432?

X9? 5.7900.187?

X10??? 4.180

X11??? 3.930

X12??? 5.170

X13???0.600

X14??? 2.350

X1X20.373???

X1X3à0.102 4.5600.392?

X1X4à0.052 5.9200.339?

X1X5à0.044à1.170à0.561?

X1X6?à1.730à0.488?

X2X4à1.030???

X2X7? 5.490à0.228?

X2X8? 3.0300.103?

X3X4à0.789???

X4X50.178???

X10X12???à1.350

X10X14???à1.280

X1X2X4à0.117???

X1X3X40.686???

X1X4X5à0.014???

?These terms do not take part in relevant model equations that determined for each of three consecutive experimentations,separately.

Some lipases possess a unique activating feature at the interface between an aqueous and an organic phase(Lu et al.,2007).Hence, the in?uence of water content on biodiesel conversion was studied and it was found that the effect of water on biodiesel yield was insigni?cant as compared to other variables.

3.4.Validation of all DoE experiments

Regression models were obtained separately for each response from DoE results on each of the three consecutive stages(Table5).

It was observed that there is a respectable correlation between experimental and model results for each of three experimental steps.Correlations of0.9995,0.9999,and0.9946were obtained for MPPM synthesis,MPPB synthesis,and biodiesel production, respectively.Root mean squared error(RMSE)values were 0.011MPa,0.013%,0.001U/g disk,and0.16%for mechanical strength,immobilization ef?ciency,immobilized lipase activity, and biodiesel production,respectively.

In order to validate DoE sets,an experiment for each DoE set was conducted.The accuracy of each model was validated with triplicate experiments under the maximized reaction conditions calculated using the model equations obtained.The results were given in Table6.

As seen,model results were in good agreements with the exper-

imental ones,especially for biodiesel production with an absolute error of0.45%.As a result,the model was accurate and reliable for predicting the process yields.

3.5.Effects of vegetable oils on enzymatic biodiesel synthesis

It has been reported that all the triglyceride sources should be considered as lipase substrates.Various oils(sun?ower,soybean, restaurant grease,waste vegetable oils,and animal fats)have been commonly used in enzymatic biodiesel synthesis.However,such substances as phospholipids and waxes in oils may negatively af-fect enzyme activity(Salis et al.,2007).

Three different oils(sun?ower,soybean,and waste cooking oil) were used for biodiesel synthesis by MPPB in order to compare the effectiveness of the transesteri?cation reaction.All reactions were performed for5h at65°C by using a stoichiometric excess amount of methanol(Fig.5).The highest yield was obtained from sun-?ower oil(63.8%).Biodiesel yields from soybean and waste cooking oil were55.5%and50.9%,respectively.3.6.Effects of MPPB con?gurations on enzymatic biodiesel synthesis

The in?uences of MPPB con?gurations on biodiesel synthesis from sun?ower oil were studied using MPPB in different forms (monolithic,powdered,and bead)in5h and24h reactions.In5h reaction,biodiesel yields were63.8%,81.1%,and86.9%using mono-lithic,bead,and powdered MPPB,respectively.In24h reaction,bio-diesel yields were90.2%,93.9%,and97.0%using monolithic,bead, and powdered MPPB,respectively.As can be seen,the most effective biocatalyst was powdered MPPB for the production of biodiesel.This could be due to ef?cient mixing of reactants during reaction.

4.Conclusion

This study deals with the production of biodiesel by enzymatic transesteri?cation using microporous polymeric biocatalyst (MPPB).In the study,Taguchi methodology was used for MPPM synthesis in monolithic form,lipase immobilization onto mono-lithic MPPM and?nally biodiesel production using monolithic

Table6

Levels of variables for validation of each DoE set and the estimated model results together with experimental ones.

Variables MPPM synthesis

(Mechanical strength,MPa)MPPB synthesis Biodiesel production

(Methyl esters yield,%) (Immobilization ef?ciency,%)(Lipase activity,U/g disk)

Levels Results Levels Results Levels Results Levels Results

Exp.Model Absolute

Error(%)Exp.Model Absolute

Error(%)

Exp.Model Absolute

Error(%)

Exp.Model Absolute

Error(%)

X1+1+1+1

X2à1à1à1

X3à17.127.27 2.03à1à1

X4+1+1+1

X5à1à186.1980.33 6.80à17.217.72 6.60

X6à1à1

X7+1+1

X8+1+1

X9+1+1

X10+1

X11+1

X12+159.8360.100.45 X13à1

X14+1

?Each experiment was repeated in triplicate and the results were given as arithmetical averages of the measurement values belonging to each process.

1990N.Dizge et al./Bioresource Technology100(2009)1983–1991

MPPB.In addition,MPPM in powder and bead forms were obtained and used in biodiesel production.A microporous polymeric matrix (MPPM)was synthesized using styrene,divinylbenzene,and polyglutaraldehyde.SEM micrographs and FTIR spectrum showed that copolymer can be produced as a porous structure having alde-hyde functional groups.The polymeric matrix could be prepared in a short time in any desirable shapes and is a favorable immobiliza-tion support for lipase from https://www.wendangku.net/doc/5b8453491.html,nuginosus.The enzyme was cova-lently attached onto MPPM with80%,85%,and89%immobilization ef?ciencies using bead,powder,and monolithic forms,respec-tively.It was shown that all forms of MPPB(monolithic,bead, and powder)were used successfully for the production of biodiesel using sun?ower,soybean,and waste cooking oil.The best biodiesel yields were obtained from sun?ower oil(97%).The biodiesel yield from waste cooking oil was lower(90.2%).This could be due to contaminants formed in waste cooking oil affecting the enzyme. Bead or powdered MPPB catalyzed transesteri?cation reactions re-sulted in higher biodiesel yields due to ef?cient mixing of reactants during reaction.This study shows that lipase immobilization onto newly synthesized microporous support is rather effective process in enzymatic transesteri?cation for biodiesel production.It was shown that immobilized lipase retained its activity during10re-peated batch reactions at25°C,each lasting24h.Since the devel-oped novel method is simple yet effective,it could have a potential to be used industrially for the production of chemicals(fatty acid production,removal of fatty acids from oil and production of mod-i?ed oils)requiring immobilized lipases.

Acknowledgement

This study was supported by the TUBITAK,the Scienti?c and Technological Research Council of Turkey(Project No.MAG-261). References

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