Evaluation of reaction mechanisms and the kinetic parameters for the

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Evaluation of reaction mechanisms and the kinetic parameters for the transesterification of castor oil by liquid enzymes Thalles Allan Andrade, Massimiliano Errico, and Knud Villy Christensen Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b02285 • Publication Date (Web): 07 Aug 2017 Downloaded from http://pubs.acs.org on August 8, 2017

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Industrial & Engineering Chemistry Research

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Evaluation of reaction mechanisms and the kinetic parameters for the

2

transesterification of castor oil by liquid enzymes

3

Thalles A. Andrade,* Massimiliano Errico, Knud V. Christensen

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Department of Chemical Engineering, Biotechnology and Environmental Technology, University of

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Southern Denmark, Campusvej 55, 5230, Odense M, Denmark

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*Corresponding author: [email protected]

1 ACS Paragon Plus Environment


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Abstract:

2

The use of liquid enzymes for the production of biodiesel as an alternative to chemical catalysts

3

requires significant investigation due to the lack of experimental data for the various feedstock and

4

catalyst combinations. In this paper, reaction rates and kinetic modeling of the transesterification of

5

castor oil with methanol using the enzyme Eversa® Transform as catalyst were investigated.

6

Reactions were carried out for 8 hours at 35 °C with: an alcohol-to-oil molar ratio equal to 6:1, a 5

7

wt% of liquid enzyme solution and addition of 5 wt% of water by weight of castor oil. From the

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concentration data, four different reaction mechanistic models were compared to determine the

9

mechanism that best fitted the experimental data. Mechanisms where the methanolysis and

10

hydrolysis reactions occurred simultaneously in the system were best at describing the

11

concentration profiles. The high methanolysis rates of glycerides obtained, indicated that

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transesterification dominates over hydrolysis. The mechanism among the four models proposed that

13

gave the best fit could be simplified, eliminating the kinetic parameters with negligible effects on

14

the reaction rates. This model was able to fit the experimental data at different reaction

15

temperatures.

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Keywords: biodiesel; castor oil; kinetic modeling; liquid enzyme


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Notations

2

[DAG]

Diglycerides concentration [mol L-1]

3

[E]

Free enzyme concentration [mol L-1]

4

[E ∙ i]

Enzyme complexes concentration [mol L-1]

5

[ET]

Total enzyme concentration [mol L-1]

6

[FAME]

Fatty acid methyl esters concentration [mol L-1]

7

[FFA]

Free fatty acids concentration [mol L-1]

8

[Gly]

Glycerol concentration [mol L-1]

9

k

Reaction rate constant [h-1 or L mol-1 h-1]

10

Ki

Equilibrium constants [L mol-1]

11

KMeOH

Methanol inhibition constant [mol L-1]

12

[MAG]

Monoglycerides concentration [mol L-1]

13

[MeOH]

Methanol concentration [mol L-1]

14

[TAG]

Triglycerides concentration [mol L-1]

15

Ve,FFA

Maximum forward rate constant for esterification [L mol-1 h-1]

16

V-e,FFA

Maximum reverse rate constant for esterification [L mol-1 h-1]

17

Vh,j

Maximum forward rate constants for hydrolysis [L mol-1 h-1]

18

V-h,j

Maximum reverse rate constants for hydrolysis [L mol-1 h-1]

19

Vt,j

Maximum forward rate constants for transesterification [L mol-1 h-1]

20

V-t,j

Maximum reverse rate constants for transesterification [L mol-1 h-1]

21

[W]

Water concentration [mol L-1]

22

i = TAG, DAG, MAG, FFA

23

j = TAG, DAG, MAG



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1. Introduction

2

Depletion of fossil fuel resources and increasing environmental concerns encourage the use of

3

biobased fuels as alternative and renewable sources of energy. Biodiesel is one such alternative fuel.

4

It is produced from fatty acid alkyl esters derived from the alcoholysis reaction of triglycerides

5

contained in vegetable oils and/or animal fats with short chain alcohols 1–3. Non-edible castor oil is

6

an attractive source of triglycerides, since this crop can be grown on marginal lands which are

7

unsuitable for food crops. In addition, as ricinoleic acid is the main fatty acid in this oil, castor oil

8

has high alcohol solubility. Besides their use as biofuel, esters of ricinoleic acid have different

9

industrial applications, such as a fuel additives, cosmetics, and surfactants 4–6. When considering a

10

feedstock for biodiesel though, the properties of the final product are of paramount importance. It

11

has to meet the international standards for biodiesel or biodiesel blends in order to be used in diesel

12

engines and it has to show reasonable storage stability. Many castor oil transesterification studies

13

are available in the literature 4,7–12. The methyl esters formed from castor oil are highly viscous 13,14,

14

which makes them unsuitable as biodiesel in pure form. However, when used in blends with other

15

less viscous biodiesels or mineral diesel methyl esters obtained from castor oil can satisfy most of

16

the quality parameters required by the biodiesel international standards (such as ASTM D6751 or

17

EN 14214) for biodiesel commercialization 10,15 and have been reported to perform well in engine

18

trials 13,16. As to storage stability, castor oil methyl esters are reported to be less prone to oxidation

19

or thermal degradation 17 than soybean or sunflower based fatty methyl esters, being only second to

20

petroleum based diesel 18. This is mainly attributed to the high content of ricinoleic acid esters

21

which are less predisposed to oxidation and polymerization than linolenic or linoleic esters as it

22

contains less unsaturated carbon bonds which are prone to oxidative polymerization 19.

23

In industrial processes, most of the transesterification reactions occur under alkali or acid catalysis.

24

Chemical-catalyzed routes generally proceed at fast reaction rates, providing high biodiesel yields


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20

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formation which reduces catalytic activity and make product separation more difficult. Traditionally

3

this problem is alleviated by an initial acid catalyzed esterification step that mainly esterifies the

4

free fatty acids followed by an alkali catalyzed transesterification step. The downstream processes

5

include neutralization of the alkali catalyst and wastewater treatment, which increase the process

6

energy consumption. This encourage the replacement of the homogenous chemical catalysts 14,21–23.

7

One such option capable of transforming both free fatty acids and glycerides into methyl esters in

8

one reaction step are lipases. These enzymes are substrate and product specific in the sense that they

9

do not catalyze any side reactions 24. They can simultaneously catalyze both transesterification of

. However, the alkali-catalyzed route is sensitivity to free fatty acids (FFA) as they cause soap

10

triglycerides and esterification of free fatty acids, and have a high activity in water-poor media.

11

Enzyme-catalyzed routes proceed under mild reaction conditions to provide easy recovery of

12

products and are not sensitive to large amounts of free fatty acids, with a minimal quantity of

13

wastewater being generated 14,25. The main disadvantages of the enzymatic processes are the slow

14

reaction rate and higher price of the enzymes compared to the chemical catalysts, resulting in higher

15

process costs. Even so, for waste oils and fats, enzyme catalyzed processes are marketed as being

16

competitive compared to chemically catalyzed processes 26. Though the prices of lipases has

17

decreased, in order to minimize the process costs, the reuse of these catalysts is important and has

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been studied frequently 2,27–31. Calabrò et al. 32 concluded that the reuse of enzymes for the

19

transesterification partially maintains the initial stability. In their study, Kalantari et al. 31 retained

20

about 55 % of their initial conversion capability after 5 times of recycling lipase from Pseudomonas

21

cepacia. Babaki et al. 2 evaluated the reuse of three different enzymes. The lipase from T.

22

lanuginosus remained stable and could be reused for 16 cycles without significant loss in activity (5

23

%). Lipases from C. antarctica and R. miehei also presented good reusability, keeping the enzyme

24

activities at 84 and 85 %, respectively, after 16 reuse cycles. Enzymes can be obtained in



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immobilized or liquid form. Even if immobilization enhance enzyme stability and ease recovery,

2

liquid enzymes result in a significant reduction of the process costs, since the costs of enzyme

3

immobilization are not required 33,34. For castor oil transesterification, the reuse of liquid enzymes

4

have been shown to result in a consistent high biodiesel yield over multiple batch runs using a

5

mixture of 50 % recovered and 50 % fresh enzymes 35. A viable biodiesel production with a smooth

6

reuse of enzymes does though require a careful reactor design.

7

A proper reactor design for biodiesel production catalyzed by enzymes requires two significant

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preliminary steps. The first step refers to the definition of the optimal reaction conditions such as

9

temperature, alcohol-to-oil molar ratio, water and enzyme solution contents, and use of solvents.

10

Due to enzyme inhibition caused by excess of alcohol, stepwise addition of the alcohol also needs

11

to be considered during this stage 29. The second step concerns the evaluation of an appropriate

12

reaction mechanism and its corresponding kinetic parameters essential to perform the optimal

13

reactor design. A previous work addressed optimizing the reaction conditions. Under optimal

14

reaction conditions, a biodiesel yield of about 94 % was obtained at 35 °C, 5 wt% of enzyme

15

solution by weight of castor oil, 5 wt% of additional water, and a 6.0 alcohol-to-oil molar ratio for

16

castor oil transesterification with methanol and the liquid enzyme Eversa® Transform as catalyst 36.

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Nevertheless, investigation of detailed reaction mechanisms and reaction kinetics for the ricinoleic-

18

methanol reaction system has not been reported yet.

19

In the preceding decades different mechanisms have been proposed for the enzymatic

20

transesterification of different vegetable oils, such as corn, palm, jatropha, and waste cooking oil,

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catalyzed by immobilized enzymes 32,37–45. Most of the models are based on Ping-Pong Bi Bi

22

mechanisms that describe the competition between the substrates and enzyme inhibition 46. Few

23

kinetic studies involving liquid enzymes for transesterification are available in the literature 34,47–50

24

and none of these investigate the kinetic mechanism for the methanolysis of castor oil catalyzed by


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liquid lipases. Detailed kinetic studies are essential for the design of biorefineries with accurate

2

values. The present study evaluates different kinetic mechanisms.

3

Eversa® Transform was used as catalyst to produce fatty acid methyl esters (FAME) from castor oil

4

and methanol. Initially, two different mechanisms were tested based on previous studies found in

5

the literature 39,49. Both these mechanisms follow Ping-Pong Bi Bi mechanism and assume that

6

some steps are irreversible. However, while the first model claims that tri-, di-, and monoglycerides

7

are hydrolyzed into FFA and then turned into biodiesel by esterification, the second assumes that

8

transesterification and hydrolysis can happen in the reaction simultaneously. As these assumptions

9

were found lacking, reversible reactions were incorporated in both kinetic mechanisms in order to

10

obtain a more precise and accurate kinetic model.



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2. Materials and Methods

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2.1 Materials

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The liquid enzyme Eversa® Transform (lipase activity 100 kLU g-1, in an enzyme mixture

4

containing 75 vol% of water and 25 vol% of propylene glycol) was kindly provided by Novozymes

5

A/S (Denmark). Ecological castor oil was purchased from Urtegaarden ApS (Denmark). The

6

following chemicals and reagents were purchased from Sigma-Aldrich, of analytical grades:

7

methanol (99.9 % purity), acetonitrile (99.9 %), hexane (97.0 %), and isopropanol (99.9 %). Fatty

8

acids methyl esters (methyl ricinoleate, methyl linoleate, and methyl oleate) and free fatty acids

9

(ricinoleic acid, linoleic acid, and oleic acid), used as HPLC standards, were also acquired from

10

Sigma-Aldrich. All the HPLC standards had a purity grade greater or equal to 99 %. Standards of

11

triglycerides (TAG), diglycerides (DAG), and monoglycerides (MAG) were subsequently prepared

12

by transesterification and hydrolysis reactions, followed by their individual separation in

13

preparative HPLC, since they were not commercially available.

14

2.2 Enzymatic transesterification of castor oil

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The transesterification of castor oil catalyzed by liquid lipase was carried out in a 250 mL two-neck

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round-bottom flask equipped with a stirrer, immersed in a thermostat silicon oil bath, and connected

17

to a water-cooled condenser. The optimal reaction conditions for the transesterification of castor oil

18

were defined based on a previous study 36. Though the optimal reaction temperature for

19

transesterification was found to be 35 °C, for the sake of completeness, the reaction was also carried

20

out at 40 and 50 °C. The reaction mixture containing 50 g of castor oil, 5 wt% of the lipase Eversa®

21

Transform solution and an additional 5 wt% of water, by weight of castor oil, was weighed in the

22

reactor and heated to reaction temperature. The reaction mixture was stirred constantly at 750 rpm.

23

The total reaction time was 8 hours. Methanol was added into the reactor as four step additions, at


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two-hour intervals, totaling 6.0 methanol-to-oil molar ratio after the last addition. Stepwise addition

2

was required to minimize enzyme inhibition by the alcohol. Samples of the reaction mixture were

3

taken at predefined reaction times, and centrifuged for 5 min at 6000 rpm in a Spectrafuge™ Mini

4

Laboratory Centrifuge to separate the biodiesel from the glycerol and the enzymatic phase. The

5

biodiesel phase was weighed and diluted to 10 mg mL-1 in a 4:5 w/w hexane to isopropanol

6

solution, mixed thoroughly with a vortex mixer, and filtered through a Whatman 0.2 µm filter unit

7

into small vials to be analyzed. All the reactions were carried out in duplicate.

8

2.3 Sample HPLC analysis

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An Agilent 1200 Series High-Performance Liquid Chromatography (HPLC) system, with UV

10

detection at 205 nm, equipped with a Phenomenex Luna C18 reverse-phase column (150 × 4.60

11

mm, particle size 3 µm) was used to determine the composition of the samples at different reaction

12

times. A three-gradient system (acetonitrile, water and a 4:5 w/w mixture of hexane–isopropanol)

13

was used as a solvent mixture at a flow rate of 0.8 mL min-1. A runtime of 35 minutes was required

14

for each analysis. The concentrations of tri-, di-, and monoglycerides, as well as the concentrations

15

of fatty acid methyl esters (biodiesel) and free fatty acids were obtained from the calibration curves

16

of each component. Concentrations of glycerol, water and methanol were calculated via a material

17

balance.

18

2.4 Kinetic modeling and data handling

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Experimental data for the reaction component concentrations as a function of reaction time were

20

collected. Based on the proposed reaction mechanisms, the model that gave the best fit to the

21

components concentration profiles had to be found. Different approaches to survey the allowable

22

parameter space for a globally optimal fit, including stochastic optimization methods have been

23

successfully applied for parameters estimation 51–53. Here, the approach chosen by Li et al.49 was



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followed. To this purpose Matlab v8.5 (The MathWorks, Inc.) was used to solve the material

2

balances and estimate the kinetic parameters of the suggested mechanisms. The function ode45 was

3

used to solve the ordinary differential equations by numerical integration, and through the

4

optimization function lsqcurvefit, the kinetic parameters were obtained by minimizing the error of

5

the nonlinear data-fitting. To this purpose, lsqcurvefit minimizes the sum of the squares of the

6

difference between the calculated value and the experimental data using the Maquard-Levenberg

7

method, leading to the statistical optimal parameter fit. The influence of the methanol stepwise

8

addition on the concentrations, at each two hours of reaction, was taken into account in the

9

mathematical modeling by calculating new concentrations of the components right after the

10

additions of methanol.


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1


3. Theory

2

The general reaction for the transesterification of TAG is given by Eq. 1. Reaction between one

3

mole of triglycerides and three moles of alcohol produces 3 moles of fatty acid alkyl esters

4

(biodiesel), and is usually described as a sequence of three reversible and consecutive steps:

5

conversion of TAG into DAG followed by DAG converted into MAG, and finally MAG into

6

glycerol 23. TAG + 3 Alcohol ⇌ 3 Biodiesel + Glycerol

(1)

7

In the presence of water, reversible triglyceride hydrolysis occurs in the system. The general

8

hydrolysis reaction is described by Eq. 2. Similarly to the transesterification, hydrolysis of one mole

9

of TAG with three moles of water is described in three consecutive steps, producing three moles of

10

free fatty acids (FFA) 54. FFA reacts with alcohol and the esterification of FFA generates fatty acid

11

alkyl acids, as shown in Eq. 3. TAG + 3 Water ⇌ 3 FFA + Glycerol

(2)

FFA + Alcohol ⇌ Biodiesel + Water

(3)

12

3.1 Enzymatic kinetics

13

The enzymatic reaction can be described by the Ping-Pong Bi Bi mechanism in which the enzyme

14

reacts with the substrate forming an active intermediate enzyme-substrate complex. This complex

15

can be decomposed back to the substrate and the free enzyme or broken down into products,

16

releasing the enzyme which is then free to form a new enzyme-substrate complex 55,56.

17

Even though the total concentration of the enzyme (ET) is usually known, the concentrations of the

18

free enzyme (E) and the enzyme associated into complexes are not easily measured. The use of the

19

pseudo-steady-state hypothesis (PSSH) overcomes this problem, since it assumes that the net rate of

20

formation of each enzyme complex is equal to zero.



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1

The presence of an inhibitor influences the rate of enzyme-catalyzed reactions. The inhibitory

2

substance competes with the substrate for the enzyme molecules to form an inhibitor-enzyme

3

complex 57 and thus reduces the rate of reaction by inhibiting enzyme access to the substrate. When

4

multiple reactants and products are involved, this relatively simple model becomes more complex.

5

3.2 Kinetic transesterification mechanisms

6

In the present paper, different kinetic mechanisms based on the works by Li et al. 49 (Mechanism A)

7

and Cheirsilp et al. 39 (Mechanism B) are proposed for the castor oil transesterification with

8

methanol in the presence of water, using a liquid lipase as catalyst. These mechanisms and

9

adaptions to them are described below. The study rested on the change in concentrations of

10

triglycerides (TAG), diglycerides (DAG), monoglycerides (MAG), fatty acid methyl esters

11

(FAME), free fatty acids (FFA), methanol (MeOH), water (W), and glycerol (Gly) at different

12

reaction times. To develop the mechanisms, some assumptions were considered. First of all, castor

13

oil contains mainly ricinoleic acid. The alcohol group present in this fatty acid results in castor oil

14

being highly soluble in alcohols. This solubility reduces the phase mass transfer limitations during

15

the transesterification to such an extent that they can be ignored 58. Secondly, the enzymatic

16

inhibition by methanol is assumed to follow a competitive inhibition mechanism. The pseudo-

17

steady-state hypothesis was used on the rates of formation of the enzymatic complexes.

18

Mechanisms A and B are described in detail in the following subsections.

19

3.2.1 Mechanism A

20

The first kinetic mechanism was adapted from the model presented by Li et al. 49, as shown in Fig.

21

1. In this mechanism, three successive hydrolysis reactions release FFA that is finally converted

22

into FAME by esterification. The mechanism includes the formation of the enzymatic complexes

23

TAG·E, DAG·E, MAG·E, and FFA·E, besides the inhibition by methanol, resulting in the complex


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1

MeOH·E. The formation reactions of the complexes are assumed to be reversible, while the

2

hydrolysis and esterification reactions are presumed to be irreversible. The total concentration of the

3

enzyme (ET) is given by E = E + TAG ∙ E + DAG ∙ E + MAG ∙ E + FFA ∙ E

4

(4)

Assuming pseudo-steady-state, the concentration of the free enzyme (E) can be obtained: E =

E 1 + !"# TAG + $"# DAG + %"# MAG +

6

constant for methanol. These constants, following the notation of the Fig. 1, are defined as: -. , -/.

$"#

%"# ,

=

and

-1 , -/1

&&"

%"#

are the equilibrium constants, and

%*+,

Where

=

$"# ,

MeOH/

5

!"#

!"# ,

&&" FFA +

=

-2 , -/2

&&"

=

-3 , and -/3

%*+,

%*+,

=

(5)

is the inhibition

-/5 . -5

7

The total concentration of the enzyme is constant and therefore included in the reaction rate

8

constants, resulting in the maximum rate of reaction for a given total enzyme concentration. Based

9

on this, the rate expressions for each substance for mechanism A are: E dTAG = −89!"# WTAG E dt

(6)

E dDAG = :89!"# WTAG − 89$"# WDAG; E dt

(7)

E dMAG = :89$"# WDAG − 89%"# WMAG; E dt

(8)

E dFAME = :8*&&" FFAMeOH; E dt

(9)

E dFFA =

Evaluation of reaction mechanisms and the kinetic parameters for the

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