4. Sugar Fatty Acid Ester Synthesis in CO 2 saturated acetone
4.2.1 Screening the reaction – First observation
Glucose, palmitic acid, enzyme and acetone were introduced into the reactor. A very small amount of glucose and palmitic acid were used for the screening process because the sampling and analysis methods to determine a glucose concentration are limited (Appendix A1).
High pressure CO2 was introduced to achieve a pressure in the middle range of the pressure of countercurrent gas extraction. Figure 4.2 shows the reaction behavior with pressure, temperature, and amount of enzyme. It is obvious that, SCCO2 is required to support the synthesis in acetone.
As is commonly known, the introduced amount of enzyme is related to the substrate concentration. A full investigation on enzyme concentration, substrate ratio, acetone concentration, temperature, and pressure was conducted in the following studies. These key process parameters were studied and a reaction mechanism was suggested. In those cases, conversion was determined by calculating the residual fatty acids instead of glucose.
0 10 20 30 40 50
50°C, 100 bar, 60mg enzyme
50°C, 100 bar, 30mg enzyme
40°C, 100 bar, 30 mg enzyme
40°C, 1 bar, 30 mg enzyme
Conversion (%)
Figure 4.2: Screening synthesis of sugar ester.4
4.2.2 BEffect of acetone concentration
Pure SCCO2 can dissolve a very limited amount of glucose [98]. Therefore, adding a polar organic solvent to improve glucose solubility is required. Acetone is selected because it is accepted by EEC directives 88-344-CEE as an extraction solvent in the manufacturing of food products and additives [140]. The phase distribution in the reactor is dependent on the amount of acetone introduced into the reactor. With a small amount, acetone is totally dissolved in SCCO2. An exceeding amount of acetone leads to an expanded saturated liquid mixture, here CO2
saturated acetone [47, 141]. Figure 4.3 shows the initial reaction rate vs. the acetone concentration. At 3% acetone (Vacetone/Vreactor), there is a maximum, where the reaction takes place in the expanded liquid phase. An increased amount of acetone at a constant substrate quantity dilutes the substrate concentration and slows down the reaction. The reaction is not
4 40 mL acetone, 2h, 4 mg palmitic acid, 2 mg glucose, Novozyme 435
favored at an acetone level of 2% or less because acetone and CO2 form a gaseous phase, which cannot dissolve much glucose. A good contacting of substrates and the active site of the enzyme is required. The best condition is that the reaction takes place in the expanded liquid phase. Pfohl et al. [52] reported that CO2 does not dissolve more than 3% of common cosolvents at pressures below 10 MPa. Therefore, no significant glucose concentration can be expected in the vapor phase.
0 200 400 600 800 1000 1200
2% 3% 4% 6% 8%
Acetone concentration [Vacetone/Vreactor]
Initial rate [μmol/(genzyme.h)] BFigure 4.3: Effect of acetone concentration on the initial reaction rate.5
4.2.3 BEffect of enzyme type
Basically an enzyme is a protein, so its structure is strongly affected by the physicochemical properties of the reaction media. Especially in presence of acetone, the intra- molecular hydrogen bonds and hydrophobic associations in the lipase can be disrupted by the intermolecular hydrogen bond formation such as (lipase)-N-H-..O=C(CH3)2 (acetone), leading to a drastic change in the lipase’s conformation [92]. To improve the stability of the enzyme, different immobilization methods have been applied. As a result, each type of immobilized enzyme has a specific characteristic for reaction and ability to withstand the reaction condition. In
5 P=65bar, T=50°C, 50mg palmitic acid, 35mg glucose, 15mg Novozyme 435
this study, some common types of immobilized enzymes have been tested to catalyze esterification of glucose and palmitic acid. This screening process was performed in order to select the best enzyme suitable for the work. The result in Figure 4.4 shows that among the investigated enzymes, Novozyme 435 is the best, maintaining a high activity in a wide range of temperature. Lipozyme RM IM only catalyzes well at an optimum temperature of 50°C.
However, the initial rate of this enzyme is rather low. Lipozyme TL IM could not be used for this type of esterification of glucose palmitate in acetone under high pressure CO2. Only a very low initial rate was detected with this enzyme at 40°C, and no reaction was observed at a higher temperature. The behavior of these enzymes obtained in this work is similar to previous studies in other reaction media reported by [88, 101, 142] .
0 200 400 600 800 1000
40 50 60
Temperature [°C]
Initial rate [μmol/(genzyme.h)]
novozyme 435 Lipozyme RM IM Lipozyme TL IM
Figure 4.4: Screening of different lipases for sugar ester synthesis.6
4.2.4 BEffect of enzyme concentration
In general, sugar is only slightly soluble into high pressure CO2-acetone [52]. As a result, despite an exceeding amount of glucose is initially being loaded into the reaction vessel, the concentration of glucose in the reaction medium is kept constant at a certain temperature and pressure. Therefore, esterification depends on the amount of fatty acid dissolved in the mixture.
6 P=65bar, 50mg palmitic acid, 35mg glucose, 15mg enzyme, 20mL acetone
Figure 4.5 shows the conversion depending on the amount of enzyme, based on the amount of dissolved palmitic acid. It is observed that first the amount of enzyme increases the total conversion, since more active sites are available for the reaction. However, at higher loadings of the enzyme, the reaction slows down because of agglomeration of the enzyme particles, which lowers their activity and decreases reaction rate and conversion. The optimum amount of enzyme was recorded at 30% Novozyme 435 related to the amount of dissolved palmitic acid. The fatty acid loading capacity of CO2 expanded acetone will be presented in section 3.2.1.
0 3 6 9 12 15
0 10 20 30 40 50 60
Enzyme concentration related to palmitic acid [%]
Conversion [%]
BFigure 4.5: Effect of Novozyme 435 concentration on the conversion of palmitic acid.7
4.2.5 BTemperature effect
Temperature affects the esterification of glucose and palmitic acid. It is observed that the conversion increases with temperature (Figure 4.6). Conversion at 50 or 60°C is better than the one at 40°C. However, there is no significant difference between the conversion obtained at 50°C and 60°C. At higher temperature, the internal energy and the average distance of the substrate molecules are increased, leading to reduction of the inter-hooking of the substrate molecules. On the other hand, the effect of pressure and temperature are related [1]. Changing temperature will also change the solubility of glucose and palmitic acid. Beside that, temperature has an effect on
7 P=65bar, T=50°C, 2h, 50mg palmitic acid, 35mg glucose, 20mL acetone
the configuration of the active site of the enzyme. With this type of high pressure CO2-acetone system, Novozyme 435 has an optimum stability temperature in the range of 50 to 60°C.
0 5 10 15 20 25
0 50 100 150 200 250 300
Reaction time [min]
Conversion [%]
40°C 50°C 60°C
BFigure 4.6: Temperature effect on the conversion of palmitic acid.8
4.2.6 BPressure effect
The effect of pressure on the esterification reaction is shown in Figure 4.7. It is observed that the reaction could not take place without applying SCCO2. At atmospheric pressure, there is almost no reaction found at 40°C and a very low conversion at 60°C. When CO2 is introduced into the system, from up to 65 bar, the reaction is accelerated. This can be explained by effect of high pressure CO2 on the viscosity and the phase distribution of the system. Viscosity will be strongly reduced when the organic solvent is saturated by SCCO2 [141]. Mass transfer is therefore improved. More substrates are transported to the active sites of the enzymes, speeding up the conversion rate. Phase distribution is well defined under CO2 pressure. The amount of glucose and water can be distributed between light and heavy phases of a high CO2 pressurized acetone system [52]. As a result, the level of water bound around the active site of the enzyme can be controlled by the pressure applied. However, pressure has also a strong effect on the volume expansion [143-145]. An increase in pressure increases the volume of the reaction
8 P=65bar, 50mg palmitic acid, 35mg glucose, 15mg Novozyme 435, 20mL acetone
mixture and reduces the concentration of the substrate. In this study, a pressure of 65 bar is suggested because there is no significant difference between the reaction rate at 65 and 105 bar.
0 200 400 600 800 1000 1200
1 65 85 105
Pressure [bar]
Initial rate [μmol/(genzyme.h)]
40°C 60°C
BFigure 4.7: Effect of pressure on the initial reaction rate.9 4.2.7 BMolar ratio effect
The synthesis reaction is also affected by the substrate ratio. It is known that a high initial amount of sugar will inhibit the conversion to sugar ester [146]. Therefore, a slight solubility of sugar in the CO2 saturated acetone may have an advantage from this point of view. However, it is required to contact both substrates with the active site of the enzyme for the catalytic reaction.
The substrate ratio can be varied, depending on physico-chemical properties. Figure 4.8 shows the influence of the molar ratio from 1:1 to 1:8 of glucose to palmitic acid. The initial reaction rate increases with a molar ratio up to 1:2. A further increase in molar ratio reduces the reaction rate. Compared with the reaction at atmospheric pressure, this optimum substrate ratio shifts towards a little more fatty acid content. Tarahomjoo et al. esterified palmitic acid with glucose and observed that conversion increased with increase in molar ratio of glucose from 0.5 to 1 and there was no further increment beyond 1:1 of glucose to palmitic acid [88].
9 50mg palmitic acid, 35mg glucose, 15mg Novozyme 435, 20mL acetone
The behavior as observed in Figure 4.8 can be explained by the effect of substrate molar ratio to the substrate solubility and viscosity of the reaction mixture. When the palmitic acid molar is changed, the molar partial ratios of glucose and even the acetone and water in the CO2
expanded phase are also influenced. Therefore, in CO2 saturated acetone, the increase of the amount of palmitic acid to glucose can be attributed to a decrease in the amount of dissolved glucose. Less glucose substrate available for the absorption onto the active sites of the enzyme will slow down the reaction rate. Moreover, the amount of fatty acid has also an effect on the viscosity. The higher the input of palmitic acid is, the higher is the viscosity. For this reason, lower reaction rates were observed at molar ratios higher than 1:2.
0 200 400 600 800 1000 1200
1:1 1:2 1:4 1:6 1:8
Molar ratio (glucose : palmitic acid) Initial rate [μmol/(genzyme.h)]
BFigure 4.8: Effect of substrate molar ratio on the initial reaction rate.10
4.2.8 BEffect of adding water
The effect of water on the esterification was investigated by introducing various amounts of water to the reaction medium. As shown in Figure 4.9, the initial reaction rate could be increased up to 25 % when 0.5 % (Vwater/Vacetone) water was introduced into acetone initially.
However, after that point, the larger the amount of added water was, the lower was the reaction rate. As commonly known, a mono hydration layer at the surface of the enzyme is required for the catalyst activation. Because acetone is a polar solvent, water is stripped off from the enzyme
10 P=65bar, T=50°C, 20mL acetone, 30% Novozyme 435
surface. As a result, adding a small amount of water can compensate this effect and accelerate the reaction. However, adding too much water will shift the equilibrium of the reaction towards the hydrolysis of the ester, resulting in low yields of the synthesis. Excess water can also lead to agglomeration of the enzyme, decreasing the surface area available for the reaction [34]. It was also reported that as a product of the esterification reaction, water affects not only the enzyme activity but also the thermodynamics of the equilibrated reaction [147].
0 200 400 600 800 1000
0 1 2 3 4 5 6
Added water [Vwater/Vacetone, %]
Initial rate [μmol/(genzyme.h)] BFigure 4.9: Effect of additional water on the initial reaction rate.F11
Based on the data in Table 2.4, section 2.2.5, originally reported by Pfohl et al [52], for the applied reaction conditions, the maximum amount of water dissolved in the lighter phase is less than 3.75 %. The excess water will be transferred into the heavy phase, and the lighter phase will be saturated with water. Therefore, the water content in the lighter phase is kept constant, controlled by the temperature and pressure applied. This is a very important point, because during the synthesis reaction, a continuous removal of the water generated during the reaction, is necessary. Adding of molecular sieves to adsorb water is a choice for an investigation. Chaiyaso studied the esterification of palm fatty acid distillates with glucose in acetone [86]. It was found that no product could be detected in the absence of molecular sieves in the reaction mixture. In
11 P=65bar, T=50°C, 50mg Palmitic acid, 35mg glucose, 15mg Novozyme 435, 20mL acetone
Water saturated
this study, the enzyme was introduced at the bottom of the reactor. In case of water in excess, part of the enzymes was directly contacted to the heavy phase (water phase). The backward hydrolysis was accelerated, decreasing the forward ester synthesis rate. However, if we could design a system, in which the enzyme is isolated in the upper part of the lighter phase, the water could be removed out of the enzyme active site. This is the starting point to suggest a reaction mechanism for a continuous reaction, which will be presented in the following part.
4.2.9 BReaction mechanism
Based on the phase diagram reported by Pfohl et al. [52], as re-presented in Figure 4.10, a reaction mechanism is suggested as shown in Figure 4.11. The reaction is performed in a heterogeneous system, where most of the glucose is not dissolved, but suspended in the reaction mixture. A pseudo dynamic equilibrium between glucose, palmitic acid, glucose palmitate, acetone, CO2, and H2O is proposed. Solid glucose will be continuously dissolved in the light phase (enriched acetone and CO2), converted into glucose palmitate through enzymatic reaction.
Glucose palmitate will be precipitated as a solid, if its concentration increases beyond the solvent capacity of the expanded liquid mixture. Water as a by-product is transferred to the water phase.
By this way, excess water can be removed without using any water removal tools, such as adding a molecular sieve or using distillation.
Figure 4.10: VLLE in the glucose + acetone + water + CO2 system. T = 60°C, experimental data (dashed lines, ▼- 4.16 MPa, ■ - 6.07 MPa, ● - 8.23 MPa) and calculations (solid lines) with the Soave-redlich-Kwong EOS [52].
BFigure 4.11: Reaction mechanism for esterification of palmitic acid and glucose in a high pressure acetone-CO2 system.
The advantage of using the shifting of an equilibrium in this multiphase system can be compared to the biphasic organic-water system, demonstrated by Bommarius [148]. The substrate and product of the enzymatic conversion are strongly dependent on the shifting of equilibrium between the heavy phase (L1) and the light phase (L2).
We symbolize the reaction A B in such a system above.
PA and PB are defined by Equation 4.4 as partition coefficients in phase L1 and L2.
L21 L
A A
P A and
L21 L
B B
P B (4.4)
In both L1 and L2 phase, equilibrium exists between A and B
11
1
L L
L A
K B and
22
2
L L
L A
K B . (4.5)
A total equilibrium constant for both phases L1 and L2 could be defined as:
Total Total Total
A
K B . (4.6)
There is a relation between concentration of A, B in phase L1, L2 and total concentration as follows:
ATotalVTotal AL1VL1 AL2VL2 (4.7)
BTotalVTotal B L1VL1 BL2VL2 (4.8)
2
1 L
L
Total V V
V (4.9)
Combining Equation 4.7 & 4.8 and introducing into Equation 4.6, we obtain:
L11 L11 L22 L22 L L L L Total
V A V A
V B V K B
(4.10)
A new form of KTotal in Equation 4.12 is reached when it is divided by AL1VL1 and the phase ratio
2 1
L L
V
V
introduced. (4.11)
A B L
Total
P K P
K
1 1
2 . (4.12)
In case the reaction is of the form A + B C + D, KTotal will have the value shown in Equation 4.13.
) 1 )(
1 (
) 1 )(
1 (
2
B A
D C
L Total
P P
P K P
K
(4.13)
In bi-phasic organic solvents, the total equilibrium constant KTotal is set up for a specific reaction condition. However, in CO2 saturated acetone that is a dynamic value. Because the phase ratio changes as water is released during the reaction, KTotal will be not constant but time dependent. Besides, KTotal is a strong function of pressure and temperature, which affect substrate distribution in the light and heavy phases. For this complicated behavior of the reaction kinetics of this system, it is recommended that experiments are carried out for every individual substrate of the esterification to obtain the optimum condition.
4.2.10 BReaction progress and reaction kinetics
For an enzymatic synthesis in the batch mode, reaction time is an important factor. It is determined by the resistance of adsorption and desorption between the enzyme, substrates and products. SCCO2 in the near critical region was proven to enhance the activation of the enzymes, and create favorable concentrations of substrate in the microenvironment of the enzyme [149].
With CO2 saturated acetone, reaction improvement has also been proven through this study as mentioned in the previous parts. Therefore, the reaction time required for the synthesis is
shortened, compared to the traditional reaction in acetone without using high pressure CO2. Figure 4.12 shows that the reaction obtains a steady state after ca. 6 hours. There was no significant difference in conversion after 6 hours and 18 hours, which can be attributed to the degree of saturation which decreases in this batch mode. At normal pressure, a couple of days are required for the synthesis to reach steady state. Chiaiyaso et al. [86] studied biocatalytic acylation of carbohydrates with fatty acids from palm fatty acids distillates (PFAD). A conversion of 76%
glucose ester was obtained from esterification of glucose and PFAD by Novozyme 435 in acetone at 40°C after 74 hours. Arcos et al. also studied the esterification of glucose with fatty acids using Novozyme 435 and acetone at 40°C and 60°C [78]. They discovered that the conversion increased with increase in temperature and obtained a stable conversion after 72 hours.
To describe the progress curve, a first-order exponential increase in product concentration was used. The details of this kinetics are described in the previous section 2.2.2. Figure 4.12 shows that the experimental data are correlated well with this first-order exponential approach (Appendix B1). The product is expressed as the amount of reacted palmitic acid instead of product concentration. The reaction at 50 °C is much faster than the one at 40 °C, which starts very slowly with a delay of ca. 1hour, corresponding to the high melting point of palmitic acid.
0 1500 3000 4500 6000
0 4 8 12 16 20 24
Time (h) Reacted palmitic acid (Umol/g enzyme)
50°C 40°C
Figure 4.12: Reaction progress curve at different temperature.F12
12 P=65bar, 100mg palmitic acid, 35mg glucose, 30mg Novozyme 435, 20mL acetone