Project Based Assignments Article Review on Reaction Kinetics

Priyanka Tiwari Feb 27th 2012 ChE 218

Z. D. Knezevic, S. S. Marinkovic and L. V. Mojovic, Kinetics of Lipase-Catalyzed Hydrolysis of Palm Oil in Lecithin/Isooctane Reversed Micelles, Applied Microbiology Biotechnology, 49, 267-271, 1998.

CONTENTS 1 2 Purpose of Study...................................................................................................................... 2 Methods (Mathematical and Experimental Approach) ........................................................... 2 2.1 2.2 2.3 2.4 2.5 3 4 Summary .......................................................................................................................... 2 Assumptions and Variables .............................................................................................. 2 Mathematical Methods employed .................................................................................... 2 Experimental Methods employed..................................................................................... 2 Experimental and Theoretical Results.............................................................................. 3

Conclusions ............................................................................................................................. 4 Evaluation of the study ............................................................................................................ 4 4.1 4.2 4.3 4.4 Was the study worth doing? ............................................................................................. 4 Analysis of the experimental data .................................................................................... 5 Credibility of findings ...................................................................................................... 5 Are conclusions reasonable? ............................................................................................ 6

ii

Purpose of Study The purpose of the study is to develop a mathematical model for hydrolysis of palm oil in

order to study kinetics involved in the process.

2 2.1

Methods (Mathematical and Experimental Approach) Summary

The article selected discusses the kinetics involved in the process of hydrolysis of palm oil by using an enzyme called Candida rugosa lipase. Hydrolysis of oil leads to the production of fatty acids and glycerol. Fatty acids act as a feedstock for several chemicals such as lubricants, soaps, etc. These are also used as raw materials for food and cosmetic products. The authors have used lecithin/isooctane reversed micellar scheme for extraction. This set up results in easy separation of products from the enzyme. Because of its application in food industry, the hydrolysis utilizes natural micellar system called lecithin which is non-toxic in nature. 2.2 Assumptions and Variables

The researchers have made the following assumptions for the process: The reaction kinetics of the process follows Michaelis-Menten mechanism. E+S ES E + P. (1)

Therefore we get the following relationship v vm

m

(2)

where, v is the reaction rate of product formation , vm is the maximum reaction rate molmin-1mg-1, [S] is substrate concentration mol/dm3, Km is called as MichaelisMenten constant (mol/dm3).

The values of Km and vm are estimated using the Lineweaver-Burk Plot (obtained from linear transformation of Michaelis-Menten equation).

It is also assumed that the reaction is first order, single substrate reversible kinetic model. Therefore the reaction mechanism can be represented as: S+W P + Q where, (3)

[S] is concentration of palm oil (ester bonds), [W] is the concentration of water, [P] and [Q] are concentrations of products. 2.3 Mathematical Methods employed The authors obtained the following equation for degree of hydrolysis of palm oil. [ Where [(
( ( ) )

] )

(4) (5) ( )] is the maximum degree of hydrolysis, is the time (6)

(
)

Where X is the degree of hydrolysis,

when X reaches one half of the maximum degree. 2.4 Experimental Methods employed The researchers have derived a mathematical model for hydrolysis of palm oil, shown in Equation 4.The authors performed palm oil hydrolysis in laboratory to obtain experimental data. The reaction was carried out as a batch process. 100 cm3 of bottles were filled with 12 cm3 of lecithin/organic solution containing varying amount of substrate concentration (palm oil). Two organic solvents were tested e.g., iso-octane and n-hexane. The concentration of substrate was varied from 0.059mol/dm3 to 0.480mol/dm3. To start the reaction, buffer solution of lipase with

pH 7.0 was added to the reaction mixture. The process was carried out at a fixed temperature of 37C. The degree of hydrolysis was calculated by finding the acid value and saponification value of the oil in the reaction mixture. (7) Following parameters were obtained with respect to time: 2.5 Degree of hydrolysis Vs. reaction time Formation of products ( Free fatty acids) Vs. reaction time

Experimental and Theoretical Results

The experimental results show that the kinetics of the hydrolysis follows Michaelis-Menten kinetics for low initial substrate concentrations [ ]. It was found that for high [ ] (for e.g., 0.746 mol/dm3), lower reaction rates were obtained. Theoretical results (obtained from mathematical model, Equation 4) predicted a higher rate for high [ ]. Therefore the data didnt fit the model equation well at large values of substrate concentrations (Figure 1).

Figure 1. Shows the deviation of experimental and theoretical data at high substrate concentration (0.480 dm3). 3

Conclusions Parameters such as

m

vm were obtained from Lineweaver-Burk plot. The values of vm

m

were 1.29 mol min-1mg-1 and 1.02 mol min-1mg-1 and the values of

were 0.243 mol/dm3

and 0.6 mol/dm3 for isooctane and n-hexane respectively. As the authors obtained a low value of
m for

isooctane system, they inferred that use of lecithin isooctane solvent increased the enzyme

affinity for the substrate during reaction. Thus it was considered as more efficient solvent for the process. The authors concluded that the experimental results of the process fitted the model equation well for low values of [ ]. The discrepancy at large concentrations was explained as follows : Substrate inhibition resulting in inhibited enzyme kinetics. In this case, the basic Michaelis-Menten equation doesnt hold true. Due to the reduction in size of micellar radius. Due to deactivation of enzyme during the course of the reaction.

4 4.1

Evaluation of the study Was the study worth doing? The study was worth doing because of following reasons: The adopted method of hydrolysis was energy efficient method because of the use of enzymatic splitting of palm oil by lipase. The process occurred at low temperatures and thus it was more energy efficient. Thermal degradation of the products was reduced as the reaction was enzyme catalysed. The process was economical as the enzyme, lipase, was available at low cost. This was because the microorganisms producing the enzyme could be genetically manipulated. 4

The process was environment friendly since it required mild operating temperature, i.e., 37C that prevented enzyme denaturation. Thus the overall process cost was reduced.

The use of reverse micellar system for the process made the process energy efficient. This set up required less energy for increasing interfacial area to perform hydrolysis.

4.2

Analysis of the experimental data The authors have provided data on degree of hydrolysis versus reaction time at varying initial

substrate concentration [

] . There is no information though on the repeated runs performed for

obtaining this data. The plot of degree of hydrolysis versus temperature shown in Figure 1 follows Michaelis-Menten kinetics consistently at different lower initial substrate concentrations [ ]. Thus the data looks good. However at high range of substrate concentration [ ], deviations from experimental results are observed. The error in model is due to the simple assumptions taken to select Michaelis-Menten mathematical model. We can get rid of the error by including effects of substrate inhibition. Reaction mechanism in Equation (1) can therefore be modified as: E + S ES E + P + S (8)

ES2 v vm
m [ ]

(9)
[ ][ ] [ ]

Where

[ ][

(10)

4.3

Credibility of findings Analysis of results can be summed up as: The authors have made highly simplistic assumptions. The model is not in a generalized form. The authors have assumed that the process of hydrolysis follows single substrate, first order kinetics as depicted by Michaelis-Menten. A more generalized form includes the effect of substrate inhibition that can validate for the error in the model as shown in Equation 9. The values of Km and vm were estimated using Lineweaver Burk plot. The plot is a linearized form of Michaelis-Menten equation. It is known that linearization adds a lot of error to a model. Therefore the value of Km obtained is erroneous. Authors have not discussed the effect of agitation speed of water bath on the hydrolysis rate. Also the effect of varying pH, temperature, enzyme activity and concentration of reversed micellar solution (surfactant) are not analyzed to determine the degree of hydrolysis.

4.4

Are conclusions reasonable? First the researchers have concluded that iso-octane is a better solvent than n-hexane since Km

has a low value for iso-octane than n-hexane. The value of Km obtained is however suspicious because of the use of Lineweaver Burk plot for its determination. Secondly, the authors suspect substrate/product inhibition as a reason for deviation of experimental data from MichaelisMenten kinetics. This is fairly reasonable stated proposition. Finally the authors have also proposed enzyme deactivation as a probability for this deviation. Enzymes denaturation is result of high temperatures and fluctuating pH conditions. Since the process is carried out at a low temperature (37C) and optimum pH (pH 7.0), the proposition doesnt sound appropriate. 6