CLO3

Reactions in transesterification process

There is a consensus on the reaction steps involved in the transesterification process of triglycerides,
which are indicated in Figure 1. These are the three consecutive and reversible reactions.

Triglyceride (TG) + Methanol Diglyceride (DG) + R1-COO-CH3

Diglyceride (DG) + Methanol Monoglyceride (MG) + R2-COO-CH3

Monoglyceride (MG) + Methanol Glycerol (GL) + R3-COO-CH3

Figure 1 : Reversible reaction steps in the formation of methyl esters from triglycerides.

In the first reaction, triglyceride (TG) molecule reacts with methanol to produce diglyceride (DG) and
a fatty acid methyl ester (R1-COO-CH3). Then, in the second reaction, diglyceride reacts with
methanol to form monoglyceride (MG) and another molecule of fatty acid ester (R 2-COO-CH3). In the
third reaction, monoglyceride (MG) reacts with alcohol to produce glycerol (G) and a third molecule
of fatty acid ester (R3-COO-CH3).

Enzyme kinetics

The reduction in methanol concentration in the experiments was an indication for biodiesel
production, so the bioreactor proved its suitability for synthesising biodiesel. In addition, the
effectiveness of the enzyme used in this work contributes significantly to biodiesel formation. In this
case, Novozyme 435 (Candida antartica B, immobilized on macroporous acrylic resin) is used to
synthesize biodiesel.The enzyme substrate mechanism can be explained by the following reaction
mechanism in Figure 2:

E+S ES P+E
Figure 2: The enzyme substrate mechanism

In this mechanism, the substrate S will bind with the enzyme E to form enzyme-substrate complex,
ES. The process of this bond formation is commonly referred to as enzymatic (or protein)
crosslinking (Heck, Faccio, Richter, & Thöny-Meyer, 2013; Jus et al., 2011). The complex is then
adsorbed by the surface of the enzyme for the reaction to take place. The product is then released
from the enzyme while the enzyme is preserved for more substrates to bind in.

It must be noted that enzymes act apparently as adsorbents, similar to those of porous carbon,
activated carbon, carbon nanotubes, fullerene, rich husk and waste carbon (obtained from fertilizer
plant, rubber tire, sugar industry etc) and thereby hypothetically the enzyme-substrate reaction
mechanisms should follow the adsorption-desorption phenomena (Ahmaruzzaman & Gupta, 2011;
Gupta, Jain, Ali, Chandra, & Agarwal, 2002; Gupta, Nayak, Agarwal, & Tyagi, 2014; Gupta et al., 2016;
Saleh & Gupta, 2014). It also has been proven that Michaelis-Menten model represents the enzyme-
catalysed reaction mechanisms perfectly (Gunawan, Suhendra, Rizkia, & Hasanah, 2017).

Kinetic Study

In this study, we developed a packed bed reactor system for transesterification of waste cooking
palm oil. The present work was focused on the reaction parameters that affected lipase-catalysed
transesterification of waste cooking palm oil with methanol using tert-butanol as solvent in a
continuously packed bed reactor.

The substrate mixture typically comprised of waste cooking oil and methanol with 1:4 mol ratio.
Tert-butanol was used as the solvent with 1:1 tert-butanol to waste cooking oil volume ratio.
Substrate flow rate and packed bed height (immobilized enzyme quantity) were varied to study the
effect of changing reactor column condition on transesterification of waste cooking palm oil. The
reaction was carried out at 40 C. Samples were collected after 3 hours of reaction time.

Parametric study

Table 1 shows the compilation of optimum condition data from experimental studies conducted in
batch system.

Parameter Optimum condition

Enzyme Novozyme 435
Solvent Tert-butanol
Solvent to oil volume ratio 1:1
Methanol to oil molar ratio 4:1
Temperature 40 oC
Table 1

Effect of temperature

Although transesterification reaction can be performed at room temperature, the process is strongly
influenced by the reaction temperature. Generally, the ideal reaction temperature is often near the
boiling point of alcohol. However, the reaction can be carried out at different temperatures,
depending on the physical and chemical properties of the oil used [13]. Several studies reported that
50–70 oC would be the best temperature range to obtain the highest biodiesel yield [28]. However,
transesterification process with Novozyme 435 should not be carried out above 50 oC because the
stability and the usage life of lipase may be affected. In this case, 40 oC is the optimum temperature
for the reaction.
Effect of enzyme

The amount of enyme is also dependent on the type of oil used in the transesterification process.
Enzyme activity increases with increasing content. Thus, a 25% enzyme load is the most feasible to
obtain the highest production of biodiesel yield.

Furthermore, lipases especially Novozyme 435 are considered to be the type of enzymes which are
able to hydrolyse fat. They play a significant role in digestion and breaking the triglycerides in oils
and lipids in the majority of living organisms. Lipases are classified according to their source of origin,
such as animals, plants or microorganisms and their properties differ accordingly.

Effect of alcohol-to-oil ratio

Transesterification reactions are reversible. Thus, excess alcohol is needed to shift the reaction
toward the forward direction [27]. Therefore, the alcohol-to-oil ratio is one of the most sensitive
factors that affect the final biodiesel yield [96]. For this reaction, in the presence of lipase enzyme,
the optimum methanol-to-waste cooking oil molar ratio is approximately 4:1.

Kinetic model

The strategies above allowed the use of the Michaelis-Menten kinetic model to estimate the kinetic
constants, Vmax and KM separately evaluating the effect of different concentrations of methanol
and WFO in the enzymatic transesterification. 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 using Equation 1.

v max [S]
v= (Equation 1)
[ S ]+ K m
where υ 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 (mol L−1), and [S] is substrate concentration
which is methanol (mol L−1).