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ENZYMATIC HYDROLYSIS OF VEGETABLES OILS BY LIPASE F-AP15 Rhizopusoryzae IN CONTINUOUS STIRRED TANK REACTOR (CSTR)

Adilene Lares-Molina * , Esther Carrillo-Prez, Manuel Prez-Tello y Juan Antonio Noriega-Rodrguez Engineering Sciences: Chemical Engineering, Chemical Engineering and Metallurgy department, Universidad de

Sonora, Blvd. Luis Encinas y Rosales S/N, Col. Centro, Hermosillo, Sonora, 83000, Mxico.*[email protected], [email protected].

Keywords : enzymatic, hydrolysis, oil, lipase

AbstractIn order to determinate the feed and output flows in a continuous stirred tank reactor (CSTR) in a

pilot plant scale (50 L), were determined hydrolysis kinetic parameters of analytic grade olive oiland commercial oil (canola, soy, sunflower and safflower mix) on an automatic titrationequipment Titralab 856. Were studied the effect of temperature (25-40C) and the concentrationsof enzyme (0.25-0.75% w/w of oil). Hydrolysis greater than 25 C was observed and that thelipase F-AP15 Rhizopus oryzae has not specificity on the type of fatty acids in oils.

IntroductionHydrolysis of oils consists of a reaction between a triacylglycerol molecule with three watermolecules, where the triacylglycerol ester bond breaks to form three fatty acids and a glyceride.In industries is used to obtain fatty acids to produce drugs, modified fats in food and cosmetics[1]. The used of enzymes, as catalysts, in this type of processes has been increased in the lastyears, because they offered greater advantages compared with other type of catalysts andreagents, although their obtaining from various sources is costly [2]. Among the advantages ofused enzymes are: moderated reaction conditions of temperature and pressure, higher specificityto substrates, reused, ease of recovery, if used immobilized way.

CSTR reactors, used for the type of reactions, have some particular advantages on fixed bed

reactors, inasmuch as it has lower construction costs and efficient agitation removed the presenceof concentration gradients and/or temperature, such, components concentrations are equal onoutputs and mass tanks fluid.

Objective of this work was to determine kinetics parameters of enzymatic hydrolysis ofvegetable oils needed to design industrial reactors.

Materials and MethodsLipase F-AP15 Rhizopus oryzae (Amano Enzyme Inc.) was evaluated in different enzymeconcentrations (0.25-0.75% w/w oil) and temperature (25 and 40C) for oil hydrolysis of oliveoil analytic grade (Sigma-Aldrich) and commercial oil (canola, soy, sunflower and safflower

mix).

Experimental design. All experiments were made in 100 mL glass jacket reactors (Celstir,Wheaton) maintain temperature with a recirculated water bath (Figure 1). Reaction mixture was

prepared homogenizing 40% v/v of phosphate buffer (0.1 M, pH 7.0). Initiating reaction, previously dissolved lipase in 3 mL of buffer was added to the reaction mixture, according to theexperimental design (Table 1). 24 hours reaction is conducted where was added KOH 0.1 needed


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to neutralized free fatty acids in mixture and maintain a pH 7.0 using an automatic titrationstation Titralab 856 (radiometer Analytical) according to a agitation rate of 850 rpm.

For the experimentation was used the pre-determinate method OQ Method 2 PID, this pH-Stat program has a PID controller.

Table 1. Experimental design for vegetable oil hydrolysis by lipase F-AP15.

Figure 1. Continuous stirred tank reactor experimental mounting analyzed by pH-Stat

Results Analysis. Free fatty acids (FFA) obtained by reaction were calculated AOCS method 5a-40 [3]. Were determinate kinetic parameters k 0, k cat, K M, V max and catalytic efficiency using a

pseudo-first deactivation order model (eq. 1) [1] and Michaelis-Menten integrated equation (eq.2) [4].

[ ]

( )

Where: k 0, is a constant for hydrolysis initial rate ( mol FFA / mol oils); k D, is deactivation constant(s-1); V max, reaction rate when lipase is saturated with substrate ( mol/s); K M , Michaelis-Mentenconstant ( mol/L); [S 0] and [FFA] were initial substrate and free fatty acids concentrationrespectively ( mol/L).

To determine constants using previously models, Excel Solver package 2007 was used, usingmathematical methods that permitted a correlation of R2=1.0.

Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5Substrate Olive Olive Olive Commercial CommercialEnzyme concentration (%w/w oil) 0.25 0.75 0.75 0.25 0.75Vol. Substrate (mL) 24 24 12 24 12Vol. Buffer (mL) 36 36 18 36 18Temperature (C) 25 25 40 25 40


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In practice, a continuous reactor is operated in stable state conditions, in other words, withoutreagent accumulation or products in reactor, therefore, the expression used for this kind ofreactor is the following, which assume the simple Michaelis-Menten kinetic.

(

)

Where: D is the dilution rate (s -1) which is equal to the relation of caudal and reaction volumemixture; X, is substrate to product conversion.

For reactor design has established 60 mL of reaction mixture as initial volume to scalar to a 50liters pilot plant, which is going to be in a continuous adding of reagents necessary. Flows weredeterminate based in mass balance for a continuous reactor (eq. 3), and experimental results were

by establishing input reagents flows and output products flows [5].

ResultsFigure 2 shows hydrolysis progress for both oils in function of time. Pseudo-first order modelapplied fit correctly (continuous lines) experimental data (symbols) for all experiments(R 20.98).

Figure 2. Kinetics of enzymatic hydrolysis of olive oil (a) and commercial oil (b) by lipase F-AP15.

Was observed an increase in conversion and reaction rate when was increase enzymeconcentration (Exp. 1 & 2) 0.25% to 0.75% w/w oil, but was no proportional to amount ofenzyme aggregate. Increasing temperature (25 to 40C) activity and conversion were decreasing(Exp. 1 & 3;4 & 5), as reported by Ben Salah (1994), this happen due microorganism nature

where was the enzyme origin [6].

In Figure 3 shows experimental data linearization (eq. 2) to obtain kinetic parameters, whereslope is K M and ordinate in origin is V max . There was not observed significant variations on slopevalues, meaning that enzyme doesnt show specificity for mono and polyunsaturated fatty aWas observed a good adjustment to experimental data (R2>0.99).

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 5 10 15 20

H y

d r o

l y s i s

( [ F F A ] / [ S

0 ] )

Time (h)

A). Substrate: Olive oil

Exp. 1

Exp. 2

Exp. 3

Exp 1. [ E ]0 = 0.25%p/p ; 25C

Exp 2. [ E ]0 = 0.75%p/p; 25 C

Exp 3. [ E ]0 = 0.25%p/p; 40 C

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 5 10 15 20

H y

d r o

l y s i s

( F F A ] / [ S

0 ] )

Time (h)

B). Substrate: Commercial oil

Exp. 4

Exp. 5

Exp 4. [ E ]0 = 0.25%p/p; 25 C

Exp 5. [ E ]0 = 0.75%p/p; 40 C


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There was not observed significant differences on conversion and reaction rate, although fattyacids composition in oils differs between substrates, where oleic acid concentration

predominates in olive oil and linoleic acid on commercial oil.

Figure 3. Michaelis-Menten integrated equation to determine kinetic parameters.

In Table 2 resumes determinate kinetic parameters to fit mathematical models. With CSTR mass balance (eq. 3) calculated input and output flows, considering maximum conversion obtained byexperiments and calculated kinetic constants. Last column shows input and output flows for thisreaction in 50 liters CSTR.

Table 2. Kinetic parameters and necessary flows for enzymatic hydrolysis for vegetables oils by lipase F-AP15 incontinuous reactor (CSTR).

ConclusionThe mathematical models used to determinate kinetic parameters fit appropriately toexperimental data (R 2>0.98). Models are appropriate for this kind of reactions due kinetic

behavior analysis from the beginning to the reaction end.

When was greater the enzyme concentration, although, the rate and conversion percent wasincreased, there wasnt proportional to the amount of released enzyme. Thereforerecommendable to increase enzyme amount where is a long time reaction.

Was observed temperature effect on reaction, concluding that optimal temperature was at 25C.Lipase didn t show specificity to mono and polyunsaturated fatty acids on oils used inexperiments. With kinetics parameters was proposed a 50 liters pilot plant continuous reactordesign.

Exp 1. y = 128.67x - 2.2546R = 0.9927

Exp 3. y = 144.94-.02595R = 0.998

Exp 2. y = 128.51x - 0.4174R = 0.9988

0

0.05

0.1

0.15

0.2

0.25

0 0.05 0.1 0.15 0.2 0.25

[ F F A ] / t

(Ln(([FFA]/[So]-[FFA])+1))/t

Exp 5. y = 144.44x - 0.1076R = 0.9997

Exp 4. y = 128.85x + 0.9216R = 0.992

0

2

4

6

8

10

12

14

16

18

20

0 0.05 0.1 0.15

[ F F A ] / t

(Ln(([FFA]/[S 0]-[FFA])+1))/t

Exp. Conversion k 0 x 105

(mol/ mol s)k cat x 10

8 (s -1)

V maxx10 8(mol/s)

K M x 103

(mol/L) K M/k cat

(mol/L s)Q x 10 (60 mL)

(m3/s)Q (50 L)

(m 3/s)1 0.6 2.46 1.624 2.98 0.129 0.357 3.2 0.272 0.7 5.38 2.113 11.6 0.129 11.62 13.8 1.153 0.2 1.72 0.125 0.69 0.145 1318 8.5 0.714 0.6 2.83 1.388 2.97 0.129 0.788 3.1 0.255 0.12 1.16 0.538 0.10 0.145 483.3 6.3 0.52


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Nomenclature

CSTR continuous stirred tank reactor (1)D Dilution rate (s -1)FFA Free Fatty Acids (1)

[FFA] Free Fatty Acids concentration ( mol/L)k cat Catalytic constant (s -1)k D deactivation constant (s -1)

K M Michaelis-Menten constant ( mol/L) k 0 constant for hydrolysis initial rate ( mol FFA /mol oils)KOH Potassium hydroxide (1)PID proportional-integral-derivative (1)rpm revolutions per minute (1)S0 initial substrate (1)[S0] initial substrate concentration ( mol/L)V max reaction rate when lipase is saturated with substrate ( mol/s)X substrate to product conversion (1)

References

Noriega, J A. Purificacin de una Lipasa de las Vsceras de la Sardina (Sardinops sagax caerulea ) y Evaluacin desu Actividad Lipoltica Sobre los cidos Grasos PoliinsaturadosTesis de Doctorado , Instituto Tecnolgico deVeracruz, 2010.1. Ramachandra , F. H. Hydrolysis of Oils by Using Immobilized Lipase Enzyme: A Review Biotechnol.

Bioprocess Eng. 7:57-66, 2002. 2. AOCS , Official Methods and Recommended Practices of the AOCS, 6th Edition. (2000).3. Bisswager, H. Enzyme Kinetics: Principles and Methods, Weinheim: Wiley-VCH, 2002.4. Gacesa, P. Tecnologia de las Enzimas. Editorial Acribia S.A, 1990. 5. Ben Salah, A. Revue fran aaise des corps gras, 41:133-137, 1994.

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