Scholarly article on topic 'Enzymatic biodiesel production kinetics using co-solvent and an anhydrous medium: a strategy to improve lipase performance in a semi-continuous reactor'

Enzymatic biodiesel production kinetics using co-solvent and an anhydrous medium: a strategy to improve lipase performance in a semi-continuous reactor Academic research paper on "Chemical engineering"

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Abstract of research paper on Chemical engineering, author of scientific article — Laura Azócar, Rodrigo Navia, Leticia Beroiz, David Jeison, Gustavo Ciudad

Enzymatic biodiesel production kinetics under previously optimized conditions were investigated. Waste frying oil (WFO) was used as the raw material, Novozym 435 as catalyst, methanol as acyl acceptor and tert-butanol as co-solvent. To investigate pure transesterification kinetics improving product properties, 3Å molecular sieves were incorporated into the reaction to provide an anhydrous medium avoiding the side reactions of hydrolysis and esterification. The effects of either WFO or methanol on the reaction rate were analyzed separately. The reaction was described by a Ping Pong mechanism and competitive inhibition by methanol. The results obtained in the kinetics study were applied in the operation of a semi-continuous reactor for biodiesel production. The operational conditions of each reaction cycle were: methanol-to-oil ratio 8/1 (mol/mol), 15% (wt) Novozym 435, 0.75% (v/v) of tert-butanol, 44.5°C, 200rpm and 4h of reaction time. The enzymes were successively reused by remaining in the reactor during all the cycles. Under these conditions, biodiesel production yields higher than 80% over 7 reaction cycles were observed. Both the kinetics study and the reactor operation showed that Novozym 435 was not inhibited at high methanol concentrations and that the kinetics of the proposed enzymatic process could be comparable to the conventional chemical process.

Academic research paper on topic "Enzymatic biodiesel production kinetics using co-solvent and an anhydrous medium: a strategy to improve lipase performance in a semi-continuous reactor"

Accepted Manuscript

Title: Enzymatic Biodiesel Production Kinetics Using Co-solvent and an Anhydrous Medium: A Strategy to Improve Lipase Performance in a Semi-continuous Reactor

Author: Laura Azocar Rodrigo Navia Leticia Beroiz David Jeison Gustavo Ciudad

PII: DOI:

Reference:

S1871-6784(14)00048-X http://dx.doi.Org/doi:10.1016/j.nbt.2014.04.006 NBT 692

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Received date: Revised date: Accepted date:

22-11-2013

23-4-2014 25-4-2014

Please cite this article as: Azocar, L., Navia, R., Beroiz, L., Jeison, D., Ciudad, G.,Enzymatic Biodiesel Production Kinetics Using Co-solvent and an Anhydrous Medium: A Strategy to Improve Lipase Performance in a Semi-continuous Reactor, New Biotechnology (2014), http://dx.doi.org/10.1016/j.nbt.2014.04.006

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Enzymatic Biodiesel Production Kinetics Using Co-solvent and an Anhydrous Medium: A Strategy to Improve Lipase Performance in a Semi-continuous Reactor

1 12 1 12 12 Laura Azócar , Rodrigo Navia ' , Leticia Beroiz , David Jeison ' , Gustavo Ciudad '

Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Casilla 54-D, Temuco, Chile1

Departamento de Ingeniería Química, Universidad de La Frontera, Casilla 54-D, Temuco, Chile2

laura.azocar@ufrontera.cl

rodrigo.navia@ufrontera.cl

leticiaberoiz@gmail.com

david.jeison@ufrontera.cl

gustavo.ciudad@ufrontera.cl

Corresponding author: Laura Azocar Phone: ++56 45 2734195 Fax: ++56 45 2325053 e-mail: laura.azocar@ufrontera.cl

Keywords

Biodiesel; Kinetic Parameters; Waste Frying Oils; Transesterification; Bioreactors, Enzyme Biocatalysis

Highlights

■ Strategy to study only transesterification kinetic in enzymatic process to biodiesel production

■ The strategy allowed high enzyme tolerance to methanol up to 8/1 (methanol-to-oil molar ratio)

■ Kinetics of enzymatic process investigated was comparable to conventional chemical process

■ The strategy allowed enzymes reutilization in a semi-continuous reactor without activity loss

■ Semi-continuous reactor in cycles of 4 hours of reaction time reached stable FAME production of 80%

Abstract

Enzymatic biodiesel production kinetics under previously optimized conditions were investigated. Waste frying oil (WFO) was used as the raw material, Novozym 435 as catalyst, methanol as acyl acceptor and tert-butanol as co-solvent. To investigate pure transesterification kinetics improving product properties, 3A molecular sieves were incorporated into the reaction to provide an anhydrous medium avoiding the side reactions of hydrolysis and esterification. The effects of either WFO or methanol on the reaction rate

were analyzed separately. The reaction was described by a Ping Pong mechanism and

competitive inhibition by methanol. The results obtained in the kinetics study were applied in the operation of a semi-continuous reactor for biodiesel production. The operational conditions of each reaction cycle were: methanol-to-oil ratio 8/1 (mol /mol), 15% (wt) Novozym 435, 0.75% (v / v) of tert-butanol, 44.5°C, 200 rpm and 4 hours of reaction time. The enzymes were successively reused by remaining in the reactor during all the cycles. Under these conditions, biodiesel production yields higher than 80% over 7 reaction cycles were observed. Both the kinetics study and the reactor operation showed that Novozym 435 was not inhibited at high methanol concentrations and that the kinetics of the proposed enzymatic process could be comparable to the conventional chemical process.

1. Introduction

Kinetic studies of fatty acid alkyl ester (FAAE) production using different lipases have been reported in the literature. The early studies on this topic focused on free fatty acid (FFA) esterification for FAAE production. Krishna and Karanth [1] investigated the lipase-catalyzed esterification kinetics of FFA using the immobilized lipase Lipozyme IM-20 from R. miehei. The reaction mechanism was described by a Ping Pong Bi Bi model including competitive inhibition by the two substrates (isoamyl alcohol and butyric acid).

Similarly, Al-Zuhair et al. [2] studied butyric acid esterification catalyzed by a lipase from

Mucor miehei using methanol as acyl acceptor. The results fitted to a Ping Pong mechanism model, including methanol competitive inhibition.

Although interesting results have been reported regarding FAAE production by esterification, industrial interest is focused mainly on the production of FAAE from triacylglycerides (TG), and not particularly from FFA [3]. Therefore, recent kinetic studies using lipases have focused on FAAE production from TG, such as synthetic waste frying oil [4] and crude palm oil [5]. According to Talukder et al. [5], FAAE production from TG can be carried out by lipases through two consecutive reactions of hydrolysis and esterification. In these successive reactions, TG are first hydrolyzed to produce FFA and subsequently FFA are esterified to produce FAAE. Alternatively, Al Zuhair et al. [3] reported that FAAE production could occur by a direct transesterification of TG. Similarly, Cheirsilp et al. [6] established that the concept of hydrolysis and transesterification occurring simultaneously is more appropriate than the model of two consecutive reactions of hydrolysis and esterification.

According to the previous analyses, kinetic studies showing the performance of lipase

catalyzed processes for FAAE production have been already reported. However, the quality

of the final product was not considered. In this context, Azocar et al. [7] showed that when

Candida antarctica lipase immobilized on acrylic resin (Novozym 435) was used in an

anhydrous organic medium, fatty acid methyl ester (FAME) production occurred mainly by

the transesterification pathway, avoiding hydrolysis and esterification reactions [7]. When

FAME is mainly produced by hydrolysis and esterification, the final product is always

characterized by a high acid value due to FFA produced during hydrolysis reaction [8]. In

contrast, if FAME is mainly produced by transesterification reaction in anhydrous medium,

the acid value of the produced biodiesel is near to that established by the different

international biodiesel standards and additionally, and it is possible to decrease the content of intermediary products, such as mono- and diglyceride [7]. Therefore, it is of interest to study transesterification reaction kinetics by using an anhydrous medium to produce biodiesel in a lipase-catalyzed process. In addition, an anhydrous medium would allow us to investigate pure transesterification kinetics for FAAE production using lipase as the catalyst.

On the other hand, several efforts have been made in both enzyme reutilization and enzyme activity enhancement. In this sense, the use of the moderate polar co-solvent tert-butanol improves the miscibility between the alcohol and vegetable oil, increasing the mass transfer, and with the added benefit of promoting a high lipase activity and enzyme reuse [7, 9-10]. However, in previous kinetic studies, non-polar co-solvents such as n-hexane [34, 11] and n-hexadecane [12] have been predominately used. In addition, using polar co-solvents was not effective to achieve high lipase activities [13]. Regarding raw materials, transesterification kinetics using alternative feedstock, such as waste frying oil (WFO) [14], have been investigated only by Al-Zuhair et al. [4]. Therefore, there is a need to use both appropriate co-solvents and alternative raw materials in kinetic studies of FAAE production using lipases.

The aim of this work was to study FAME production kinetics mainly through the transesterification pathway in an anhydrous medium. The reaction was catalyzed by Novozym 435, with WFO as raw material, methanol as the acyl acceptor and tert-butanol as the co-solvent. In addition, a semi-continuous reactor for FAME production was operated under the optimal conditions established in kinetic trials.

2. Materials and methods

2.1 Materials

WFO collected from restaurants was filtered and characterized prior to its use as feedstock (Table 1). Density was measured at 20 °C using a manual densimeter. Kinematic viscosity was measured at 40 °C using a capillary viscosimeter. The acid value was determined by titration with KOH using phenolphthalein as indicator. The peroxide value was determined by titration with Na2S2O3, and iodine by the Wijs method [15]. Candida antarctica lipase immobilized on acrylic resin (Novozym 435) donated by Novo Industries (Denmark) was used as the catalyst. Molecular sieves (3 A) used to generate the anhydrous medium and tert-butanol used as the co-solvent were from Sigma-Aldrich. Methyl heptadecanoate was used as an internal standard and was chromatographically pure. All other chemicals were of analytical grade.

2.2 Determination of enzymatic transesterification kinetic constants

The effect of alcohol and WFO in the transesterification reaction were evaluated separately to obtain the following kinetic constants: the maximum reaction rate Fmax (mol L-1 min-1), the dissociation constants for WFO (W) and methanol (M), respectively KW and KM (mol L-1), and the inhibition constant for methanol KiM (mol L-1).

2.2.1 Reaction conditions

Each experiment on FAME production to determinate the kinetic constants was carried out in 25 mL Erlenmeyer flasks with ground glass stoppers to avoid methanol loss. The reaction conditions were established according to Azocar et al. [7]: 15% Novozym 435 (% wt based on oil weight); 44.5 °C; 0.75% (v/v) of tert-butanol as co-solvent, 0.5 g of 3 A

molecular sieves with mixing at 200 rpm during 4 hours of reaction time. Twenty four experiments were carried out, and each was repeated four times.

Samples of 50 |iL were taken every 5 min during the first 30 min of reaction time. After that, samples of the same volume were taken every 30 min, until completing 4 h reaction time. The samples were analyzed to determine FAME yield by gas chromatography.

2.2.2 Effect of methanol concentration on enzymatic transesterification

To determine the effect of methanol concentration in transesterification kinetics, experiments were run using methanol concentrations between 100 - 3,000 mol L-1. The initial WFO concentration (300 mol L-1) was chosen to avoid limiting and inhibitory concentrations according to previous experiments (data not show).

For each experiment the final concentration was defined according to the total reaction volume of WFO and methanol added to the reaction. The methanol-to-oil molar ratio ranged between 0.6/1 and 15/1.

2.2.3 Effect of WFO concentration on enzymatic transesterification

To determinate the effect of WFO concentration in transesterification kinetics, experiments

were run using WFO concentrations between 200 and 350 mol L-1. The initial methanol

concentration was chosen to avoid limiting and inhibitory concentrations according to

results obtained when effect of WFO was investigated (1,400 mol L-1).

For each experiment, the final concentration was defined according to the total reaction

volume of WFO and methanol added to the reaction. The methanol-to-oil molar ratio

ranged between 3/0.375 and 3/1. The higher dosage of WFO was established according to

the minimal quantity necessary to reach the stoichiometric relationship that would allow a complete transesterification reaction.

2.2.4 Determination of Vmax, KM, KW and KIM

The strategies above allowed the use of the Michaelis - Menten kinetic model to estimate the kinetic constants, Vmax, KM, KW and KIM, separately evaluating the effect of different concentrations of methanol and WFO in the enzymatic transesterification. The data for product concentration versus the initial reaction time (20 minutes) were plotted for each methanol or WFO concentration evaluated. The initial reaction time was established considering a slope with R > 0.9.

To determine Vmax and KM, data of the initial reaction rate obtained for different methanol concentrations were analyzed by means of a Lineweaver-Burk plot (or double reciprocal plot) (Eq 1). The same procedure was performed to determine Vmax and KW.

1 - Km • 1 .+_ 1

" Vmax [M] Vmax (1)

Where u is the initial reaction rate (mol L-1 min-1), Vmax is the maximum reaction rate (mol L-1 min-1), KM is the dissociation constant for methanol (M) (mol L-1), and [M] is methanol concentration (mol L-1).

The obtained values were then optimized using Excel solver to find the minimum objective function (Eq. 2) that compares the measured reaction rate with that predicted by the proposed kinetic equation. The inhibition constant for methanol KiM was first assumed according to previous reports [4] to be subsequently optimized by Excel solver.

OF = X(upred - u exp)2

Where OF is the objective function, u pred the predicted rate of reaction and u exp the experimental rate of reaction.

2.3 Kinetic model

According to the results obtained in the determination of the kinetic constants, a Ping Pong model with competitive inhibition by methanol with respect to the WFO was shown to best describe the reaction according to the following equation:

[M] (3)

Where u is the initial reaction rate (mol L-1 min-1), Vmax is the maximum reaction rate (mol L-1 min-1), KW and KM are the dissociation constants for WFO (W) and methanol (M), respectively (mol L-1), [W] is WFO concentration (mol L-1), [M] is methanol concentration (mol L-1) and KiM is the inhibition constant for methanol (mol L-1).

2.4 Reactor for FAME production by transesterification

A semi-continuous reactor was designed for FAME production mainly by transesterification reaction (Figure 1). The reactor vessel was glass with 0.5 L reaction volume. A glass heating jacket with hot water controlled by thermostat was used for

temperature control and a magnetic stirrer for stirring. A packed column filled with 20 g of 3 A molecular sieves was connected to the reactor to continuously remove the water from the reaction. The reaction mixture was continuously recirculated through the column to produce and maintain anhydrous conditions. To complete the set-up, a glass settler was connected to the reactor to separate the products after the transesterification reaction. The reactor was used to carry out successive reactions of FAME production. For the startup, 15.3 g Novozym 435 (15% wt based on the feedstock oil weight), 83 mL of tert-butanol (0.75% v/v), 110 mL of WFO and 41 mL of methanol (methanol-to-oil molar ratio 8/1) were added to the reactor. The total volume of the mixture inside the reactor was 234 mL. Each reaction was carried out at 44.5 °C with stirring at 200 rpm over 4 h reaction time. Recirculation of the mixture through the molecular sieve column was continuous during the reaction. After each reaction, the mixture was transferred to the settler to separate the products. After 1 h of sedimentation, glycerol was removed from the bottom of the settler and the FAME were subsequently extracted using the same procedure and analyzed by gas chromatography.

The reaction cycles following were repeated using the same procedure described previously. Lipases added in the first cycle were kept inside the reactor during all cycles, being successively reused.

2.5 Sample analysis

The reaction samples were centrifuged for 10 minutes at 4000 rpm. The upper layer was

extracted and subsequently treated at 85 °C for 30 min to eliminate the residual solvents,

tert-butanol and methanol. FAME yield was determined by quantification of FAME

content in the treated sample, carried out using a Clarus 600 chromatograph coupled with a

Clarus 500T mass spectrometer from Perkin Elmer (GC-MS). An Elite-5ms capillary column with length 30 m, thickness 0.1 p,m and internal diameter 0.25 mm was used. Sample vials were prepared by adding 3 ^g of sample to 100 ^L methyl heptadecanoate as an internal standard (initial concentration of 1300 mg L-1). The following temperature program was used: 50 °C for 1 min and then increasing temperature at a rate of 1.1 °C/min up to 187 °C. The split vent flow rate was 50, both the injector and detector temperatures were 250 °C and He was used as the carrier gas.

3. Results and Discussion

3.1 Effect of methanol concentration on initial reaction rate

The effect of methanol concentration on FAME yield was investigated (Figure 2). The experiments were carried out at a methanol-to-oil molar ratio in the range of 0.6/1 to 15/1. According to Figure 2, two main concentration zones could be distinguished. In the lower area, FAME yields of less than 60% were obtained at methanol-to-oil molar ratios lower than the stoichiometric ratio (< 3/1 methanol-to-oil molar ratio). In the upper zone, a FAME yield higher than 60% was reached at methanol-to-oil molar ratios similar to or higher than the stoichiometric ratio (> 3/1 methanol-to-oil molar ratio). This tendency was observed up to a methanol-to-oil molar ratio of 8/1, where a FAME yield of over 90% was reached at 4 h of reaction. At the highest molar ratios investigated (methanol-to-oil molar ratio of 12/1 and 15/1) a reduction in FAME yield was observed.

These results are in accord with the findings of Phan and Phan [16] who investigated

chemical FAME production using WFO as raw material, methanol as acyl acceptor and

NaOH as basic catalyst. They reported an increment in FAME yield (88%) when the

methanol-to-oil molar ratio was increased up to 8/1. However, the yield decreased for

molar ratios higher than or equal to 12/1 (82%) in a reaction of 80 min. in another study of FAAE production using a chemical catalyst (KOH), ethanol as acyl acceptor and WFO as raw material, Encinar et al. [17] also reported a decreasing FAME yield to 66% when using a methanol-to-oil molar ratio of 15/1. Similarly, Mahamuni et al. [18] using a chemical catalyst to produce FAME in an ultrasonic reactor, reported a FAME production yield increase when the methanol-to-oil molar ratio increased from 4/1 to 6/1. In contrast, FAME production yield decreased when the ratio increased to 9/1 (mol/mol). The optimal results obtained in this work compared to previous reports using chemical catalysts could be related to the avoidance of mass transfer problems by using tert-butanol, as well as minimizing enzyme inhibition. In addition, the decreased FAME yields at high methanol levels could be attributed to the fact that a methanol excess may interfere in the separation of FAME and glycerol by increasing glycerol solubility [16]. As the molar ratio increases, so does the separation cost of methanol from biodiesel by distillation [19], and therefore a methanol-to-oil molar ratio of 8/1 was selected for further experiments. To calculate the kinetic constants, the FAME yields obtained at different methanol concentrations were expressed in FAME concentration and plotted to calculate the initial reaction rate. Figure 3 shows the linear increment in FAME concentration with the increment in the initial methanol concentration during the first 20 minutes of reaction.

3.2 Effect of WFO concentration and initial reaction rate

A non-inhibitory, non-limiting concentration of methanol was used in these assays to

evaluate the sole effect of WFO concentration on FAME yield. Experiments were carried

out at a methanol-to-oil molar ratio between 3/0.375 and 3/1. FAME yields obtained with

different WFO concentrations were expressed as FAME concentration. The data of the

product concentration versus the initial reaction time (20 minutes) were plotted for each WFO concentration. The initial reaction time was established considering a slope with R > 0.9, similar to the methanol kinetics methodology. The change in the initial reaction rate with different initial concentrations of WFO is shown in Figure 4; there was no WFO inhibition in the range of the initial concentrations investigated. Similar results of competitive inhibition by alcohol, without inhibition by substrate were reported by Al Zuhair et al. [2]. However, in another investigation by the same authors, WFO inhibition during the transesterification reaction was reported [4]. Moreover, inhibition caused by FFA has been also found [1].

In the current investigation, the observed non-inhibitory effect of WFO could be related to the anhydrous medium used to carry out the experiments. This could avoid parallel reactions of hydrolysis and esterification, thereby favoring the transesterification reaction [7]. The strategy to avoid such side reactions prevents the formation of intermediary products such as FFA. In contrast, Al Zuhair et al. [3] utilized an organic medium favoring side reactions of hydrolysis and esterification. Therefore, the inhibition they found could be related to the inhibition caused by FFA previously proposed [1].

According to [20], substrate inhibition could be produced when immobilized lipases are used, as the enzyme immobilization support could adsorb the substrate producing mass transfer limitations. However, in our current investigation, this possible limitation was avoided as Novozym 435 is a lipase immobilized in a hydrophilic material. In addition, a tert-butanol system can diminish miscibility problems in the reaction medium, increasing mass transfer and reaction performance. Therefore, the results obtained here indicate limitation by both alcohol and WFO. However, only alcohol inhibition was observed,

suggesting a Ping Pong mechanism with competitive methanol inhibition when describing the Novozym 435 transesterification kinetics.

3.3 Kinetic model for enzymatic transesterification

The proposed mechanism of FAME production in anhydrous medium using Novozym 435 and considering a Ping Pong model was based on previous studies [3,6]. The proposed mechanism considers that in an anhydrous medium FAME is mainly produced through transesterification pathway. Additionally, FFA esterification reactions are considered to occur only in a first stage. Figure 5a shows the transesterification mechanism with the enzyme (E) reacting with triacylglycerol (TG) to form the first complex (ETG), after which a diacylglycerol (DG) and an acylated enzyme-fatty acid complex are formed (EAcF). Subsequently, the complex reacts with alcohol (A) to form an acylated enzyme-alcohol complex (E-AcA). In this stage, Al-Zuhair et al [3] established that the oxygen atom from the alcohol molecule could be linked to the carbon atom of the carbonyl group of the acyl enzyme intermediate to form the complex E-Ac A. Finally, the transesterification is concluded, producing FAME and the free enzyme (E). After these stages, two additional FAME molecules are produced consecutively from diacylglyceride and monoacylglyceride, by the same mechanism.

Figure 5b shows the esterification mechanism with the enzyme (E) reacting with free fatty

acid (FFA) to form acylated enzyme-FFA complex (E Ac FFA). Subsequently, the

complex reacts with alcohol (A) to form an acylated enzyme-alcohol complex (E-AcA).

Water (W) and FAME are then produced. Although water is produced during esterification

reactions, the proposed mechanism in anhydrous medium considers that hydrolysis

reactions are avoided due to the medium utilized, where molecular sieves adsorb any water.

This means that FFA will not be produced by hydrolysis reactions and, therefore, an esterification reaction will only occur at the beginning of the process due to the FFA content of the raw oil.

The kinetic parameters were determined by using Lineweaver Burk plots. Figure 6 shows the double reciprocal plot of the study of variable methanol concentrations. Kim and Kiw were determined by using Excel solver (Eq. (3)).

Kinetic parameters obtained were compared with references showing significant differences (Table 2). Krishna and Karanth [1] used butyric acid and isoamyl alcohol with Lipozyme IM-20 as the catalyst in a n-hexane system. In this study, it was found that substrate inhibition occurred, probably because butyric acid, being a short-chain polar acid, concentrates in the microaqueous layer and causes a pH drop in the enzyme microenvironment leading to enzyme inactivation. In the present study it was possible that long chain FFA present at the beginning of the reaction (Table 1) did not produce inhibition because the water was removed during the reaction by the molecular sieves. Despite these differences, there are some similarities with other reported investigations. In the study carried out in [21], the experimental conditions were similar to the current work, and no substrate inhibition was observed. This result could be related to the use of tert-butanol as co-solvent in both cases. In addition, the results shown in Table 2 are in agreement with the higher inhibitory effect of methanol compared to WFO. In this sense, Al-Zuhair et al. [3] only found substrate inhibition at methanol-to-oil molar ratios higher than 1/4.

The results obtained for the kinetic constants, KW and KM, indicate a higher affinity for

WFO compared to methanol. The high KM value indicates the strong methanol

concentration dependence of transesterification, where high methanol concentrations are

required to increase transesterification reaction rate catalyzed by Novozym 435, avoiding substrate limitation. However, high methanol concentrations are also responsible for lipase inhibition. Therefore the use of tert-butanol as a co-solvent can avoid these problems. Although the different orders of magnitude observed in the kinetic constants showed in Table 2, a similar tendency of KM values being higher than KW values can be observed. Several transesterification and esterification kinetic studies catalyzed by lipases using short chain alcohols have reported alcohol inhibition only, which can be described by the Ping Pong mechanism with inhibition by methanol.

According to Figures 7 and 8, the Ping Pong kinetic model adequately predicted reaction performance, indicating that all simplifications assumed when carried out to use the Michaelis - Menten kinetics were suitable for kinetic constant determination. At higher methanol concentrations, the model predicted a moderate reduction in initial reaction rate. The reason for the high enzyme activity at high methanol concentrations could be related to the use of the co-solvent in the reaction. The possibility of using high methanol dosages when an enzymatic catalyst is used in biodiesel production could promote a more competitive industrial process. This is because high stoichiometric molar ratios of methanol to oil can shift the equilibrium to product formation, diminishing reaction time and therefore making a process feasible for scaling. Figure 7 shows the positive effect of increasing initial WFO concentration in the initial reaction rate.

3.4 Start up of a semi-continuous bioreactor for biodiesel production

The results described were used to establish the operational conditions of a semi-continuous

bioreactor for enzymatic FAME production (Figure 1). Novozym 435 was used as catalyst,

methanol as acyl acceptor, WFO as raw material, tert-butanol as co-solvent and molecular

sieves were used to extract water during the reaction (anhydrous medium). Methanol and WFO concentrations were established according to the results obtained in the kinetic study (methanol-to-oil molar ratio of 8/1). The operation of the reactor was carried out over 30 hours, maintaining the same enzymes inside the reactor during successive cycles at 44.5 °C, 200 rpm and 4 hours of reaction time. New doses of alcohol and WFO, as well as molecular sieves of 3 A were added in each reaction cycle.

The results during the start-up did not show any significant enzymatic activity loss during the successive reaction cycles of 4 h each one, with FAME production yields higher than 80% (Figure 9). The different starting times of each reaction show that consecutive reactions were carried out, i.e. the first from 0 to 4 hours, the second from 4 to 8 hours and so on.

The use of an anhydrous medium and tert-butanol as co-solvent are effective tools to implement a semi-continuous reactor for FAME production using Novozym 435 as catalyst. Similar results were reported in [22], where no activity loss of lipases (Novozym 435 and Lipozyme TL-IM) was found after 30 cycles of 12 hours each. In addition, FAME production yields could be improved by using a centrifugation process for glycerol and FAME separation.

The reduction in operational time of the proposed process, coupled with the possibility of

enzyme reuse during several cycles, could be economically competitive compared to a

conventional chemical process using alkaline catalyst. It has been reported that some of the

main disadvantages in lipase-catalyzed processes compared to chemical processes are the

low reaction rate, high cost and sensibility to methanol [23]. Low methanol doses are

currently used to avoid lipase-methanol inhibition, leading to methanol limitation and low

reaction rates. In the current investigation, the use of the moderate polar solvent tert-

butanol as a co-solvent allowed use of high methanol-to-oil molar ratios, avoiding lipase inhibition by methanol and, therefore, increasing the reaction rate. The reason is related to tert-butanol, which improves the solubility between the raw material and the alcohol, avoiding lipase inhibition produced by insoluble methanol in the medium [7, 10]. Regarding the high cost of lipases, in the proposed process the enzymes were successively reused without loss of activity, making the FAME to utilized enzyme ratio more favorable. The use of molecular sieves in the reaction medium allowed a first step of FFA esterification; however, after this reaction both hydrolysis and esterification were disfavored [7]. This means that transesterification under these conditions is favored, which is a faster reaction compared to hydrolysis and esterification. In contast, when WFO is used as raw material in alkaline catalytic processes, soap is produced during saponification reactions due to the high FFA content of WFO [23]. In conventional chemical processes to produce FAME from WFO, homogeneous or heterogeneous acid catalysis is used. These are energy intensive processes normally due to high reaction temperature, high alcohol to oil molar ratio and long reaction times [19]. In addition, if homogeneous acid or basic catalytic processes are used, the product must be washed to remove the residues of the chemical catalyst. This washing step is not required in the proposed process as the enzyme is a solid catalyst that can be easily recovered. Thus, the process has environmental advantages as no wastewater is generated. Finally, glycerol refining could be simplified in the proposed process as only methanol removal should be carried out. Therefore, the performance of the current investigated enzymatic process could be similar to the conventional chemical processes, without the drawbacks of chemical FAME production.

4. CONCLUSION

430 FAME production using WFO in anhydrous medium with tert-butanol as co-solvent,

431 showed no inhibition by WFO and methanol under the conditions used. Therefore, the

432 reaction was described by a Ping Pong mechanism and competitive inhibition by methanol.

433 The kinetic parameters determined were: Vmax = 0.018 mol L-1 min-1, KM, meth^ = 1030 mol

434 L-1, KW, WFO= 397 mol L-1 and KIM, methan0l= 1,815 mol L-1. Although methanol inhibition

435 occurred, it was feasible to use a high level of methanol (up to 8/1 methanol to oil molar

436 ratio), a value similar to those reported when basic catalysts are used for biodiesel

437 production. This means that under the investigated conditions enzymatic inhibition was

438 avoided.

439 In the semi-continuous reactor, enzymes were successively reused remaining in the reactor

440 during all reaction cycles. Under these conditions it was possible to maintain FAME

441 production yields of greater than 80% over 7 reaction cycles. Both the kinetic study and the

442 reactor operation showed that Novozym 435 did not present any significant activity loss at

443 high methanol concentrations. Further investigations regarding scale-up will be focused on

444 the reaction operation during a longer time period, in order to establish the enzyme's useful

445 life under the proposed conditions.

447 Acknowledgements

448 This research was sponsored by Chilean CONICYT Projects 78110106, Chilean

449 FONDECYT Projects 3120171 and 11110282, PIA Projects DI12-7001 from Universidad

450 de La Frontera.

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Figure legends

FIGURE 1 Scheme of the enzymatic bioreactor for FAME production.

FIGURE 2 FAME yields for different methanol to oil molar ratios. Operational conditions: 15% (wt) Novozym 435 (based on oil weight), 44.5 °C, 0.75% (v/v) of tert-butanol, 0.5 g of 3 A molecular sieves, 200 rpm and 12 hours of reaction time.

FIGURE 3 Determination of different reaction rates for different initial methanol concentrations using a constant initial concentration of WFO [300 mol L-1] (R > 0.9).

FIGURE 4 Correlation of the initial reaction rates and the different initial WFO concentrations.

FIGURE 5 Schematic diagram of FAME production in anhydrous medium.

FIGURE 6 Lineweaver-Burk plot of reciprocal methanol concentrations versus reciprocal initial reaction rates at fixed WFO concentrations.

FIGURE 7 Comparison between the experimental results and the Ping-Pong kinetic model equation with the estimated constants of Eq. (3) for different initial methanol concentrations and an initial WFO concentration of 300 mol L-1. ( □ ) Experimental results; (—) kinetic model curve.

FIGURE 8 Comparison between the experimental results and the Ping-Pong kinetic model equation with the estimated constants of Eq. (3) for different initial WFO concentrations and an initial methanol concentration of 300 mol L-1. ( □ ) Experimental results; (—) kinetic model curve.

FIGURE 9 FAME yield during the set-up of a semi-continuous enzymatic reactor in anhydrous medium with enzymes reutilization. Operational conditions: methanol-to-oil ratio of 8/1 (mol /mol), 15% (wt) Novozym 435 (based on oil weight), 44.5 °C, 0.75% (v/v) of fert-butanol, 200 rpm and 4 hours of reaction time.

TABLE 1

Physical properties of the WFO feedstock

Property value

Density at 20 °C ( kg/m3) 926

Kinematic viscosity at 40 °C (cSt) 47.9

Acid value (mg KOH/g) 4.6

Free fatty acid (%) 2.3

Iodine value (g I2/100g aceite) 89

Peroxide index (mEq/kg) 10.5

Samples were filtered before the analysis.

TABLE 2

Comparison between the values of Vmax, KW, KM, KiM and KiW, found in the current study with those found in previous works

Parameter References

[20] [1] [4] Current work

Vmax (mol L-1 min-1) 0.94 0.012 0.002 0.018

Kw (mol L-1) 2.61 3030 0.25 397

Km (mol L"1) 10.25 3060 0.11 1030

Kim (mol L-1) 1.60 1.05 35.0 1815

Kiw (mol L-1) - ' 6.55 28.0 -

Figures 1-9

Sedimentation pump

ieri-Butanol

Methanol

Heating jacket 44.5 °C

HI Glycerol Settler

Sampling port

Figure 1

Under stoichiometric molar ratio

Methanol to oil [mol/mol]

-•— 15 —A— 12 8.0

7.0 6.0 5.0 4.0 3.0

2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6

Time [min]

Figure 2

fail BTl 7i fii

0,06 -

0,04 -

0,02 -

Figure 3

WFO concentration, [W] [mol L-1]

Figure 4

E E-TG E-Ac-F

E-Ac-A

W FAME

E-Ac-FFA

E-Ac-A

Figure 5

700 600 500

13 400

200 100

y = 97065 x + 94.26 □

R2 = 0.962 □

0.001 0.002 0.003 0.004

1/[M] [L mol 1]

0.005 0.006

Figure 6

Methanol concentration, [M] [mol L ]

Figure 7

WFO concentration, [W] [mol L ]

Figure 8

Reaction [number]

1 2 3 4 5 6 7

Time [h]

Figure 9